AU751498B2 - flt3 ligand chimeric proteins - Google Patents

Description

0 1 V WO 98/46750 PCT/US98/07511 flt3 Ligand Chimeric Proteins The present application is a Continuation-in-Part of 08/837,026 filed April 11, 1997 which is incorporated herein by reference.

FIELD OF THE INVENTION The present invention relates to chimeric proteins or multi-functional hematopoietic receptor agonists, comprising a human flt3 agonist. These chimera proteins retain one or more activities of native flt3 ligand and the other component of the chimera protein. The chimera protein may also show improved hematopoietic cell-stimulating activity or an activity not seen for flt3 ligand and the other factor when co-administered. The chimera protein may also show an improved activity profile which may include reduction of undesirable biological activities associated with native flt3 ligand and/or have improved physical properties which may include increased solubility, stability and refold efficiency.

BACKGROUND OF THE INVENTION Colony stimulating factors which stimulate the differentiation and/or proliferation of bone marrow cells have generated much interest because of their therapeutic potential for restoring depressed levels of hematopoietic stem cell-derived cells. Colony stimulating factors in both human and murine systems have been identified and distinguished according to their activities. For example, granulocyte-CSF (G- CSF) and macrophage-CSF (M-CSF) stimulate the in vitro formation of neutrophilic granulocyte and WO 98/46750 PCT/US98/07511 2 macrophage colonies, respectively while GM-CSF and interl,eukin-3 (IL-3) have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies.

Certain factors such as flt3 ligand are able to predominately affect stem cells.

Tyrosine kinase receptors are growth factor receptors that regulate the proliferation and differentiation of a number of cell. Certain tyrosine kinase receptors function within the hematopoietic system. Flt3 (Roseate et al., Oncogene, 6:1641-1650, 1991) and flk-2 (Matthews et al., Cell, 65:1143-1152, 1991) are forms of a tyrosine kinase receptor that is related to c-fms and c-kit receptors. The flk-2 and flt3 receptors are similar in amino acid sequence and vary at two amino acid residues in the extracellular domain and diverge in a 31 amino acid segment located near the C-terminus.

Flt3 ligand is a hematopoietic growth factor which has the property of being able to regulate the growth and differentiation of hematopoietic progenitor and stem cells. Because of its ability to support the growth and proliferation of progenitor cells, flt3 receptor agonists have potential for therapeutic use in treating hematopoietic disorders such as aplastic anemia and myelodysplastic syndromes. Additionally, flt3 receptor agonists will be useful in restoring hematopoietic cells to normal amounts in those cases where the number of cells has been reduced due to diseases or to therapeutic treatments such as radiation and chemotherapy.

WO 94/28391 discloses the native flt3 ligand protein sequence and a cDNA sequence encoding the flt3 ligand, methods of expressing flt3 ligand in a host cell transfected with the cDNA and methods of WO 98/46750 PCT/US98/07511 3 treating patients with a hematopoietic disorder using flt3 jigand.

US Patent No. 5,554,512 is directed to human flt3 ligand as an isolated protein, DNA encoding the flt3 ligand, host cells transfected with cDNAs encoding flt3 ligand and methods for treating patients with flt3 ligand.

WO 94/26891 provides mammalian flt3 ligands, including an isolate that has an insertion of 29 amino acids, and fragments there of.

The human blood-forming (hematopoietic) system replaces a variety of white blood cells (including neutrophils, macrophages, and basophils/mast cells), red blood cells (erythrocytes) and clot-forming cells (megakaryocytes/platelets). The hematopoietic systems of the average male has been estimated to produce on the order of 4.5 x 1011 granulocytes and erythrocytes every year, which is equivalent to an annual replacement of total body weight (Dexter et al., BioEssays, 2;154-158, 1985).

U.S. Patent 4,999,291 discloses DNA and methods for making G-CSF the disclosure of which is incorporated herein by reference in it entirety.

U.S. Patent 4,810,643 relates to DNA and methods of making G-CSF and Cys to Ser substitution variants of G-CSF.

Kuga et al. (Biochem. Biophys. Res. Comm.

159:103-111, 1988) made a series of G-CSF variants to partially define the structure-function relationship.

Kuga et al. found that internal and C-terminal deletions abolished activity, while N-terminal WO 98/46750 PCTIUS98/07511 4 deletions of up to 11 amino acids and amino acid substitutions at positions 1, 2 and 3 were active.

Watanabe et al. (Anal. Biochem. 195:38-44, 1991) made a variant to study G-CSF receptor binding in which amino acids 1 and 3 were changed to Tyr for radioiodination of the protein. Watanabe et al. found this Tyrl, Tyr 3 G-CSF variant to be active.

Erythropoietin is a naturally-occurring glycoprotein hormone with a molecular weight that was first reported to be approximately 39,000 daltons (T.

Miyaki et al., J. Biol. Chem. 252:5558-5564 (1977)).

The mature hormone is 166 amino acids long and the "prepro" form of the hormone, with its leader peptide, is 193 amino acids long Lin, U.S. Patent No. 4,703,008). The mature hormone has a molecular weight, calculated from its amino acid sequence, of 18,399 daltons Jacobs et al., Nature 313:806-810 (1985); J. K. Browne et al., Cold Spring Harbor Symp.

Quant. Biol. 5:1693-702 (1986).

The first mutant erythropoietins erythropoietin analogs), prepared by making amino acid substitutions and deletions, have demonstrated reduced or unimproved activity. As described in U.S.

Patent NO. 4,703,008, replacement of the tyrosine residues at positions 15, 40 and 145 with phenylalanine residues, replacement of the cysteine residue at position 7 with an histidine, substitution of the proline at position 2 with an asparagine, deletion of residues 2-6, deletion of residues 163- 166, and deletion of residues 27-55 does not result in an apparent increase in biological activity. The Cys 7 -to-His 7 mutation eliminates biological activity.

A series of mutant erythropoietins with a single amino acid substitution at asparagine residues 24, 38 WO 98/46750 PCT/US98/07511 or 83 show severely reduced activity (substitution at position 24) or exhibit rapid intracellular degradation and apparent lack of secretion (substitution at residue 38 or 183). Elimination of the O-linked glycosylation site at serinel26 results in rapid degradation or lack of secretion of the erythropoietin analog Dube et al., J. Biol. Chem.

33:17516-17521 (1988). These authors conclude that glycosylation sites at residues 38, 83 and 126 are required for proper secretion and that glycosylation sites located at residues 24 and 38 may be involved in the biological activity of mature erythropoietin.

Deglycosylated erythropoietin is fully active in in vitro bioassays S. Dorsdal et al., Endocrinology 116:2293-2299 (1985); U.S. Patent No.

4,703,008; E. Tsuda et al., Eur J. Biochem.

266:20434-20439 (1991). However, glycosylation of erythropoietin is widely accepted to play a critical role in the in vivo activity of the hormone H..

Lowy et al., Nature 185:102-105 (1960); E. Goldwasser and C. K. Kung, Ann. N.Y. Acad. Science 149:49-53 (1968); W. A. Lukowsky and R. Painter, Can. J.

Biochem. :909-917 (1972); D.W. Briggs et al., Amer.

J. Phys. 201:1385-1388 (1974); J.C. Schooley, Exp.

Hematol. 13:994-998; N. Imai et al., Eur. J. Biochem.

194:457-462 (1990); M.S. Dordal et al., Endocrinology 116:2293-2299 (1985); E. Tsuda et al., Eur. J.

Biochem. 188:405-411 (1990); U.S. Patent No.

4,703,008; J.K. Brown et al., Cold Spring Harbor Symposia on Quant. Biol. 51:693-702 (1986); and K.

Yamaguchi et al., J. Biol. Chem. 266:20434-20439 (1991). The lack if in vivo biological activity of deglycosylated analogs of erythropoietin is attributed to a rapid clearance of the deglycosylated hormone from the circulation of treated animals.

This view is supported by direct comparison of the WO 98/46750 PCT/US98/07511 6 plasma half-life of glycosylated and deglycosylated erythropoietin Spivak and B.B. Hoyans, Blood 73:90-99 (1989), and M.N. Fukuda, et al., Blood 73:84-89 (1989).

Oligonucleotide-directed mutagenesis of erythropoietin glycosylation sites has effectively probed the function of glycosylation but has failed, as yet, to provide insight into an effective strategy for significantly improving the characteristics of the hormone for therapeutic applications.

A series of single amino acid substitution or deletion mutants have been constructed of erythropoietin, involving amino acid residues 15, 24, 49, 76, 78, 83, 143, 145, 160, 162, 163, 164, 165 and 166. In these mutants are altered the carboxy terminus, the glycosylation sites, and the tyrosine residues of erythropoietin. The mutants have been administered to animals while monitoring hemoglobin, hematocrit and reticulocyte levels (EP No. 0 409 113). While many of these mutants retain in vivo biological activity, none show a significant increase in their ability to raise hemoglobin, hematocrit or reticulocyte (the immediate precursor of an erythrocyte) levels when compared to native erythropoietin.

Another set of mutants has been constructed to probe the function of residues 99-119 (domain 1) and residues 111-129 (domain 2) Chern et al., Eur. J.

Biochem. 202:225-230 (1991)). The domain 1 mutants are rapidly degraded and inactive in an in vitro bioassay while the domain 2 mutants, at best, retain in vitro activity. These mutants also show no enhanced in vivo biological activity as compared to wild-type, human erythropoietin. These authors WO 98/46750 PCT/US98/07511 7 conclude that residues 99-119 play a critical role in the structure of erythropoietin.

The human erythropoietin molecule contains two disulfide bridges, one linking the cysteine residues at positions 7 and 161, and a second connecting cysteines at positions 29 and 33 Lai et al., J.

Biol. Chem. 261:3116-3121 (1986)). Oligonucleotidedirected mutagenesis has been used to probe the function of the disulfide bridge linking cysteines 29 and 33 in human erythropoietin. The cysteine at position 33 has been converted to a proline residue, which, mimics the structure of murine erythropoietin at this residue. The resulting mutant has greatly reduced in vitro activity. The loss of activity is so severe that the authors conclude that the disulfide bridge between residues 29 and 33 is essential for erythropoietin function Lin, Molecular and Cellular Aspects of Erythropoietin and Erythropoiesis, pp. 23-36, ed. I.N. Rich, Springer- Verlag, Berlin (1987)).

U.S. Patent No. 4,703,008 by Lin, F-K.

(hereinafter referred to as "the '008 patent") speculates about a wide variety of modifications of EPO, including addition, deletion, and substitution analogs of EPO. The '008 patent does not indicate that any of the suggested modifications would increase biological activity per se, although it is stated that deletion of glycosylation sites might increase the activity of EPO produced in yeast (See the '008 patent at column 37, lines 25-28). Also, the '008 patent speculates that EPO analogs which have one or more tyrosine residues replaced with phenylalanine may exhibit an increased or decreased receptor binding affinity.

WO 98/46750 PCT/US98/07511 8 Australian Patent Application No. AU-A-59145/90 by Fibi, M et al. also discusses a number of modified EPO proteins (EPO muteins). It is generally speculated that the alteration of amino acids 10-55, 70-85, and 130-166 of EPO. In particular, additions of positively charged basic amino acids in the carboxyl terminal region are purported to increase the biological activity of EPO.

U.S. Patent No. 4,835,260 by Shoemaker, C.B.

discusses modified EPO proteins with amino acid substitutions of the methionine at position 54 and asparagine at position 38. Such EPO muteins are thought to have improved stability but are not proposed to exhibit any increase in biological activity relative to wild type EPO.

WO 91/05867 discloses analogs of human erythropoietin having a greater number of sites for carbohydrate attachment than human erythropoietin, such as EPO (Asn 69 EPO (Asn 125 Ser12 7

EPO

(Thr1 25 and EPO (Pro 124 Thr 125 WO 94 /24160 discloses erythropoietin muteins which have enhanced activity, specifically amino acid substitutions at positions 20, 49, 73, 140, 143, 146, 147 and 154.

WO 94/25055 discloses erythropoietin analogs, including EPO (X 3 3 Cys 139 des-Argl 6 6 and EPO (Cys 139 des-Arg 166 Stem cell factor has the ability to stimulate growth of early hematopoietic progenitors which are capable of maturing to erythroid, megakaryocyte, granulocyte, lymphocyte and macrophage cells. Stem cell factor treatment of mammals results in absolute WO 98/46750 PCT/US98/07511 9 increases in hematopoietic cells of both the myeloid and lymphoid cells.

EP 0 423 980 discloses novel stem cell factor (SCF) polypeptides including SCF 1 48

SCF

1 15

SCF

1 16 SCF-1 61 SCF SCF SCF-- 6 2 5, SCF- 1 6 4 3, SCF 6 5

SCF

1

CF

1 85

SCF

1 188, SCF-189, SCF 122 0

SCF

1 2 4 U.S. 4,877,729 and U.S. 4,959,455 disclose human IL-3 and gibbon IL-3 cDNAs and the protein sequences for which they code. The hIL-3 disclosed has serine rather than proline at position 8 in the protein sequence.

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

Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites.

U.S. 4,810,643 discloses the DNA sequence encoding human G-CSF.

WO 91/02754 discloses a fusion protein comprised of GM-CSF and IL-3 which has increased biological activity compared to GM-CSF or IL-3 alone. Also disclosed are nonglycosylated IL-3 and GM-CSF analog proteins as components of the multi-functional hematopoietic receptor agonist.

WO 92/04455 discloses fusion proteins composed of IL-3 fused to a lymphokine selected from the group consisting of IL-3, IL-6, IL-7, IL-9, IL-11, EPO and

G-CSF.

WO 95/21197 and WO 95/21254 disclose fusion proteins capable of broad multi-functional hematopoietic properties.

GB 2,285,446 relates to the c-mpl ligand (thrombopoietin) and various forms of thrombopoietin which are shown to influence the replication, WO 98/46750 PCT/US98/07511 differentiation and maturation of megakaryocytes and megakaryocytes progenitors which may be used for the treatment of thrombocytopenia.

EP 675,201 Al relates to the c-mpl ligand (Megakaryocyte growth and development factor (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.

Summary of the Invention The present invention encompasses recombinant chimeric proteins comprising a flt3 agonist and another factor. The other factor may be a colony stimulating factor (CSF), cytokine, lymphokine, interleukin, hematopoietic growth factor which include but are not limited to GM-CSF, c-mpl ligand (also known as TPO or MGDF), M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3, IL-5, IL 6, IL-7, IL- 8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand, stem cell growth factor (SCGF) (Hiraoka, A. et al. Proc. Natl.

Acad. Sci USA 94:7577-7582, 1997) and Stromal Cellderived Factor 1 (SDF-1) (Bleul, C.C. et al., J.

Exp. Med 184:1101-1109, 1996) (herein collectively referred to as "hematopoietic growth factors". The chimera proteins can also be co-administered or sequentially administered with one or more additional colony stimulating factor(s), cytokine(s), WO 98/46750 PCT/US98/07511 11 lymphokine(s), interleukin(s), hematopoietic growth factor(s) which include but are not limited to GM- CSF, c-mpl ligand (also known as TPO or MGDF), M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, LIF, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand, SCGF and SDF-1 (herein collectively referred to "hematopoietic growth factors". These co-administered mixtures may be characterized by having the usual activity of both of the peptides or the mixture may be further characterized by having a biological or physiological activity greater than simply the additive function of the presence of the G-CSF receptor agonists or the second hematopoietic growth factor alone. The chimeric protein may also provide an enhanced effect on the activity or an activity different from that expected by the presence of the flt3 ligand or the second colony stimulating factor. The chimeric protein may also have an improved activity profile which may include reduction of undesirable biological activities associated with native human flt3.

Detailed Description of the Invention The present invention encompasses multifunctional hematopoietic receptor agonists or chimeric proteins formed from covalently linked polypeptides, each of which may act through a different and specific cell receptor to initiate complementary biological activities. Hematopoiesis requires a complex series of cellular events in which stem cells generate continuously into large populations of maturing cells in all major lineages.

There are currently at least 20 known regulators with WO 98/46750 PCT/US98/07511 12 hematopoietic proliferative activity. Most of these proliferative regulators can only stimulate one or another type of colony formation in vitro, the precise pattern of colony formation stimulated by each regulator is quite distinctive. No two regulators stimulate exactly the same pattern of colony formation, as evaluated by colony numbers or, more importantly, by the lineage and maturation pattern of the~cells making up the developing colonies. Proliferative responses can most readily be analyzed in simplified in vitro culture systems.

Three quite different parameters can be distinguished: alteration in colony size, alteration in colony numbers and cell lineage. Two or more factors may act on the progenitor cell, inducing the formation of larger number of progeny thereby increasing the colony size. Two or more factors may allow increased number of progenitor cells to proliferate either because distinct subsets of progenitors cells exist that respond exclusively to one factor or because some progenitors require stimulation by two or more factors before being able to respond. Activation of additional receptors on a cell by the use of two or more factors is likely to enhance the mitotic signal because of coalescence of initially differing signal pathways into a common final pathway reaching the nucleus (Metcalf, Nature 339:27, 1989). Other mechanisms could explain synergy. For example, if one signaling pathway is limited by an intermediate activation of an additional signaling pathway which is caused by a second factor, then this may result in a super additive response. In some cases, activation of one receptor type can induce an enhanced expression of other receptors (Metcalf, Blood 82:3515-3523, 1993) Two or more factors may result in a different pattern of cell lineages than from a single factor. The use WO 98/46750 PCTIUS98/07511 13 of multi-functional hematopoietic receptor agonists may have a potential clinical advantage resulting from a proliferative response that is not possible by any single factor.

The receptors of hematopoietic and other growth factors can be grouped into two distinct families of related proteins: tyrosine kinase receptors, including those for epidermal growth factor, M-CSF (Sherr, Blood 75:1, 1990) and SCF (Yarden et al., EMBO J. 6:3341, 1987): and hematopoietic receptors, not containing a tyrosine kinase domain, but exhibiting obvious homology in their extracellular domain (Bazan, PNAS USA 87:6934-6938, 1990). Included in this latter group are erythropoietin (EPO) (D'Andrea et al., Cell 57:277, 1989), GM-CSF (Gearing et al., EMBO J. 8:3667, 1989), IL-3 (Kitamura et al., Cell 66:1165, 1991), G-CSF (Fukunaga et al., J. Bio. Chem. 265:14008-15, 1990), IL-4 (Harada et al., PNAS USA 87:857, 1990), (Takaki et al., EMBO J. 9:4367, 1990), IL-6 (Yamasaki et al., Science 241:825, 1988), IL-7 (Goodwin et al., Cell 60:941-51, 1990), LIF (Gearing et al., EMBO J.

10:2839, 1991) and IL-2 (Cosman et al., Mol-Immunol.

23: 935-94, 1986). Most of the latter group of receptors exists in a high-affinity form as heterodimers. After ligand binding, the specific achains become associated with at least one other receptor chain (P-chain, y-chain). Many of these factors share a common receptor subunit. The a-chains for GM-CSF, IL-3 and IL-5 share the same P-chain (Kitamura et al., Cell 66:1165, 1991), Takaki et al., EMBO J. 10:2833-8, 1991) and receptor complexes for IL-6, LIF and IL-11 share a common P-chain (Taga et al., Cell 58:573-81, 1989; Gearing et al., Science 255:1434-7, 1992). The receptor complexes of IL-2, IL-4, IL-7, IL-9 and IL-15 share a common ychain (Kondo et al., Science 262:1874, 1993; Russell WO 98/46750 PCT/US98/07511 14 et al., Science 266: 1042-1045, 1993; Noguchi et al., Science 262:1877, 1993; Giri et al., EMBO J.

13:2822-2830, 1994).

The use of a multiply acting hematopoietic factor may also have a potential advantage by reducing the demands placed on factor-producing cells and their induction systems. If there are limitations in the ability of a cell to produce a factor, then by lowering the required concentrations of each of the factors, and using them in combination may usefully reduce demands on the factor-producing cells. The use of a multiply acting hematopoietic factor may lower the amount of the factors that would be needed, probably reducing the likelihood of adverse sideeffects.

Novel compounds of this invention are represented by a formula selected from the group consisting of: R1-L1-R2, R2-L1-R1, Rl-R2, and R2-R1 Where R1 is a flt3 agonist and R2 is a hematopoietic growth factor. Preferably R2 is a hematopoietic growth factor with a different but complementary activity than R1. By complementary activity is meant activity which enhances or changes the response to another cell modulator. The R1 polypeptide is joined either directly or through a linker segment to the R2 polypeptide. The term "directly" defines multi-functional hematopoietic receptor agonists in which the polypeptides are joined without a peptide linker. Thus L1 represents a chemical bond or polypeptide segment to which both R1 and R2 are joined in frame, most commonly L1 is a linear peptide to which R1 and R2 are joined by amide bonds linking the carboxy terminus of R1 to the amino terminus of L 1 and carboxy terminus of L 1 to the WO 98/46750 PCT/US98/07511 amino terminus of R2. By "joined in frame" is meant that there is no translation termination or disruption between the reading frames of the DNA encoding R 1 and R2.

A non-exclusive list of other growth factors, i.e. colony stimulating factors (CSFs), are cytokines, lymphokines, interleukins, hematopoietic growth factors which can be joined to R 1 include GM- CSF, G-CSF, c-mpl ligand (also known as TPO or MGDF), M-CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3, IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, LIF, flt3 ligand, human growth hormone, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand IL- 12. Additionally, this invention encompasses the use of modified R 1 or R2 molecules or mutated or modified DNA sequences encoding these R 1 or R2 molecules. A "c-mpl ligand variant" is defined an c-mpl ligand molecule which has amino acid substitutions and/or portions of c-mpl ligand deleted, disclosed in United States Application Serial Number 08/383,035 as well as other variants known in the art. A "G-CSF variant" is defined an G-CSF molecule which has amino acid substitutions and/or portions of G-CSF deleted, as disclosed herein, as well as other variants known in the art. Preferably, R2 is G-CSF, GM-CSF, c-mpl ligand or EPO.

The linking group (LI) is generally a polypeptide of between 1 and 500 amino acids in length. The linkers joining the two molecules are preferably designed to allow the two molecules to fold and act independently of each other, not have a propensity for developing an ordered secondary structure which could interfere with the functional t' f" WO 98/46750 PCT/US98/07511 16 domains of the two proteins, have minimal hydrophobic characteristics which could interact with the functional protein domains and provide steric separation of R1 and R2 such that R1 and R2 could interact simultaneously with their corresponding receptors on a single cell. Typically surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence. Other neutral amino acids, such as Thr and Ala, may also be used in the linker sequence.

Additional amino acids may also be included in the 41inkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the multi-functional hematopoietic receptor agonists.

Preferred L 1 linkers of the present invention include sequences selected from the group of formulas: (Gly 3 Ser)n (SEQ ID NO:1), (Gly 4 Ser)n (SEQ ID NO:2), (SEQ ID NO:3), (GlynSer)n (SEQ ID NO:4) or (AlaGlySer)n (SEQ ID NO:5), where n is an integer (collectively referred to herein as "GlySer" linkers).

One example of a highly-flexible linker is the glycine and serine-rich spacer region present within the pIII protein of the filamentous bacteriophages, e.g. bacteriophages M13 or fd (Schaller et al., PNAS USA 72: 737-741, 1975). This region provides a long, flexible spacer region between two domains of the pII surface protein. The spacer region consists of the amino acid sequence: GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGluGlyGlyGlySerGlu GlyGlyGlySerGluGlyGlyGlySerGluGlyGlyGlySerGlyGlyGlySer (SEQ ID NO:6).

WO 98/46750 PCT/US98/07511 17 The present invention also includes linkers in which an endopeptidase recognition sequence is included. Such a cleavage site may be valuable to separate the individual components of the multifunctional hematopoietic receptor agonist to determine if they are properly folded and active in vitro. Examples of various endopeptidases include, but are not limited to, plasmin, enterokinase, kallikrein, urokinase, tissue plasminogen activator, clostripain, chymosin, collagenase, Russell's viper venom protease, postproline cleavage enzyme, V8 protease, Thrombin and factor Xa.

Peptide linker segments from the hinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgE provide an angular relationship between the attached polypeptides. Especially useful are those hinge regions where the cysteines are replaced with serines. Preferred linkers of the present invention include sequences derived from murine IgG gamma 2b hinge region in which the cysteines have been changed to serines (Bell et al. US Patent 4,936,233). These linkers may also include an endopeptidase cleavage site. Examples of such linkers include the following sequences: IleSerGluProSerGlyProIleSerThrIleAsnProSerProProSerLys GluSerHisLysSerPro (SEQ ID NO:7),and IleGluGlyArglleSerGluProSerGlyProIleSerThrIleAsnProSer ProProSerLysGluSerHisLysSerPro (SEQ ID NO:8) (collectively referred to herein as "IgG2b" linkers).

The present invention is, however, not limited by the form, size or number of linker sequences employed and the only requirement of the linker is that functionally it does not interfere with the WO 98/46750 PCT/US98/07511 18 folding and function of the individual molecules of the multi-functional hematopoietic receptor agonist.

Hematopoietic growth factors can be characterized by their ability to stimulate colony formation by human hematopoietic progenitor cells.

The colonies formed include erythroid, granulocyte, megakaryocyte, granulocytic macrophages and mixtures thereof. Many of the hematopoietic growth factors have demonstrated the ability to restore bone marrow function and peripheral blood cell populations to therapeutically beneficial levels in studies performed initially in primates and subsequently in humans. Many or all of these biological activities of hematopoietic growth factors involve signal transduction and high affinity receptor binding.

Multi-functional hematopoietic receptor agonists of the present invention may exhibit useful properties such as having similar or greater biological activity when compared to a single factor or by having improved half-life or decreased adverse side effects, or a combination of these properties.

Multi-functional hematopoietic receptor agonists which have little or no agonist activity maybe useful as antagonists, as antigens for the production of antibodies for use in immunology or immunotherapy, as genetic probes or as intermediates used to construct other useful hIL-3 muteins.

The present invention also includes the DNA sequences which code for the multi-functional hematopoietic receptor agonist proteins, DNA sequences which are substantially similar and perform substantially the same function, and DNA sequences which differ from the DNAs encoding the multifunctional hematopoietic receptor agonists of the WO 98/46750 PCT/US98/07511 19 invention only due to the degeneracy of the genetic code. Also included in the present invention are the oligonucleotide intermediates used to construct the mutant DNAs and the polypeptides coded for by these oligonucleotides.

Genetic engineering techniques now standard in the art (United States Patent 4,935,233 and Sambrook et al., "Molecular Cloning A Laboratory Manual", Cold Spring Harbor Laboratory, 1989) may be used in the construction of the DNA sequences encoding flt3 ligand, EPO, G-CSF, GM-CSF, other hematopoietic growth factors and the chimeric proteins of the present invention. One such method is cassette mutagenesis (Wells et al., Gene 34:315-323, 1985) in which a portion of the coding sequence in a plasmid is replaced with synthetic oligonucleotides that encode the desired amino acid substitutions in a portion of the gene between two restriction sites.

Pairs of complementary synthetic oligonucleotides encoding the desired gene can be made and annealed to each other. The DNA sequence of the oligonucleotide would encode sequence for amino acids of desired gene with the exception of those substituted and/or deleted from the sequence.

Plasmid DNA can be treated with the chosen restriction endonucleases then ligated to the annealed oligonucleotides. The ligated mixtures can be used to transform competent bacterial cells such as E. coli strain JM101 resistance to an appropriate antibiotic. Single colonies can be picked and the plasmid DNA examined by restriction analysis and/or DNA sequencing to identify plasmids with the desired genes.

Cloning of the DNA sequences of the novel multifunctional hematopoietic agonists wherein at least one of the with the DNA sequence of the other WO 98/46750 PCT/US98/07511 hematopoietic growth factor may be accomplished by the use of intermediate vectors. Alternatively one gene can be cloned directly into a vector containing the other gene. Linkers and adapters can be used for joining the DNA sequences, as well as replacing lost sequences, where a restriction site was internal to the region of interest. Thus genetic material (DNA) encoding one polypeptide, peptide linker, and the other polypeptide is inserted into a suitable expression vector which is used to transform bacteria, yeast, insect cells or mammalian cells. The transformed organism is grown and the protein isolated by standard techniques. The resulting product is therefore a new protein which has a hematopoietic growth factor joined by a linker region to a second colony stimulating factor.

Another aspect of the present invention provides plasmid DNA vectors for use in the expression of these novel multi-functional hematopoietic receptor agonists. These vectors contain the novel DNA sequences described above which code for the novel polypeptides of the invention. Appropriate vectors which can transform microorganisms capable of expressing the multi-functional hematopoietic receptor agonists include expression vectors comprising nucleotide sequences coding for the multifunctional hematopoietic 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 multifunctional hematopoietic receptor agonist polypeptides. The vector employed in the method also contains selected regulatory sequences in operative association with the DNA coding sequences of the WO 98/46750 PCT/US98/07511 21 invention and which are capable of directing the replication and expression thereof in selected host cells.

As another aspect of the present invention, there is provided a method for producing the novel multi-functional hematopoietic receptor agonists.

The method of the present invention involves culturing suitable cells or cell line, which has been transformed with a vector containing a DNA sequence coding for expression of a novel multi-functional hematopoietic receptor agonist. Suitable cells or cell lines may be bacterial cells. For example, the various strains of E. coli are well-known as host cells in the field of biotechnology. Examples of such strains include E. coli strains JM101 (Yanish- Perron et al. Gene 33: 103-119, 1985) and MON105 (Obukowicz et al., Applied Environmental Microbiology 58: 1511-1523, 1992). Also included in the present invention is the expression of the multi-functional hematopoietic receptor agonist protein utilizing a chromosomal expression vector for E. coli based on the bacteriophage Mu (Weinberg et al., Gene 126: 33, 1993). Various strains of B. subtilis may also be employed in this method. Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. When expressed in the E. coli cytoplasm, the gene encoding the multi-functional hematopoietic receptor agonists of the present invention may also be constructed such that at the 5' end of the gene codons are added to encode Met 2 -Ala or Met at the N-terminus of the protein. The N termini of proteins made in the cytoplasm of E. coli are affected by posttranslational processing by methionine aminopeptidase (Ben Bassat et al., J. Bac. 169:751-757, 1987) and possibly by other peptidases so that upon expression WO 98/46750 PCT/US98/07511 22 the methionine is cleaved off the N-terminus. The multi-functional hematopoietic receptor agonists of the present invention may include multi-functional hematopoietic receptor agonist polypeptides having Met Ala or Met-2-Ala at the N-terminus. These mutant multi-functional hematopoietic receptor agonists may also be expressed in E. coli by fusing a secretion signal peptide to the N-terminus. This signal peptide is cleaved from the polypeptide as part of the secretion process.

Also suitable for use in the present invention are mammalian cells, such as Chinese hamster ovary cells (CHO). General methods for expression of foreign genes in mammalian cells are reviewed in Kaufman, R. 1987) Genetic Engineering, Principles and Methods, Vol. 9, J. K. Setlow, editor, Plenum Press, New York. An expression vector is constructed in which a strong promoter capable of functioning in mammalian cells drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally joined to the coding region for the multi-functional hematopoietic receptor agonist. For example, plasmids such as pcDNA I/Neo, pRc/RSV, and pRc/CMV (obtained from Invitrogen Corp., San Diego, California) can be used. The eukaryotic secretion signal peptide coding region can be from the gene itself or it can be from another secreted mammalian protein (Bayne, M. L. et al., Proc. Natl. Acad. Sci.

USA 84: 2638-2642, 1987). After construction of the vector containing the gene, the vector DNA is transfected into mammalian cells. Such cells can be, for example, the COS7, HeLa, BHK, CHO, or mouse L lines. The cells can be cultured, for example, in DMEM media (JRH Scientific). The polypeptide secreted into the media can be recovered by standard biochemical approaches following transient expression for 24 72 hours after transfection of the cells or WO 98/46750 PCT/US98/07511 23 after establishment of stable cell lines following selection for antibiotic resistance. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, Gething and Sambrook, Nature, 293:620-625, 1981), or alternatively, Kaufman et al, Mol. Cell. Biol., 5(7):1750-1759, 1985) or Howley et al., U.S. Pat. No. 4,419,446. Another suitable mammalian cell line is the monkey COS-1 cell line. A similarly useful mammalian cell line is the CV-1 cell line.

Where desired, insect cells may be utilized as host cells in the method of the present invention.

See, Miller et al., Genetic Engineering, 8:277- 298 (Plenum Press 1986) and references cited therein.

In addition, general methods for expression of foreign genes in insect cells using Baculovirus vectors are described in: Summers, M. D. and Smith, G. 1987) A manual of methods for Baculovirus vectors and insect cell culture procedures, Texas Agricultural Experiment Station Bulletin No. 1555.

An expression vector is constructed comprising a Baculovirus transfer vector, in which a strong Baculovirus promoter (such as the polyhedron promoter) drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally joined to the coding region for the multi-functional hematopoietic receptor agonist polypeptide. For example, the plasmid pVL1392 (obtained from Invitrogen Corp., San Diego, California) can be used. After construction of the vector carrying the gene encoding the multifunctional hematopoietic receptor agonist polypeptide, two micrograms of this DNA is cotransfected with one microgram of Baculovirus DNA (see Summers Smith, 1987) into insect cells, strain WO 98/46750 PCT/US98/07511 24 SF9. Pure recombinant Baculovirus carrying the multi-functional hematopoietic receptor agonist is used to infect cells cultured, for example, in Excell 401 serum-free medium (JRH Biosciences, Lenexa, Kansas). The multi-functional hematopoietic receptor agonist secreted into the medium can be recovered by standard biochemical approaches. Supernatants from mammalian or insect cells expressing the multifunctional hematopoietic receptor agonist protein can be first concentrated using any of a number of commercial concentration units.

The multi-functional hematopoietic receptor agonists of the present invention may be useful in the treatment of diseases characterized by decreased levels of either myeloid, erythroid, lymphoid, or megakaryocyte cells of the hematopoietic system or combinations thereof. In addition, they may be used to activate mature myeloid and/or lymphoid cells.

Among conditions susceptible to treatment with the polypeptides of the present invention is leukopenia, a reduction in the number of circulating leukocytes (white cells) in the peripheral blood. Leukopenia may be induced by exposure to certain viruses or to radiation. It is often a side effect of various forms of cancer therapy, exposure to chemotherapeutic drugs, radiation and of infection or hemorrhage. Therapeutic treatment of leukopenia with these multi-functional hematopoietic receptor agonists of the present invention may avoid undesirable side effects caused by treatment with presently available drugs.

The multi-functional hematopoietic receptor agonists of the present invention may be useful in the treatment of neutropenia and, for example, in the treatment of such conditions as aplastic anemia, cyclic neutropenia, idiopathic neutropenia, Chediak- WO 98/46750 PCT/US98/07511 Higashi syndrome, systemic lupus erythematosus (SLE), leukemia, myelodysplastic syndrome and myelofibrosis.

The multi-functional hematopoietic receptor agonist of the present invention may be useful in the treatment or prevention of thrombocytopenia.

Currently the only therapy for thrombocytopenia is platelet transfusion which are costly and carry the significant risks of infection (HIV, HBV) and alloimunization. The multi-functional hematopoietic receptor agonist may alleviate or diminish the need for platelet transfusion. Severe thrombocytopenia may result from genetic defects such as Fanconi's Anemia, Wiscott-Aldrich, or May Hegglin syndromes. Acquired thrombocytopenia may result from auto- or alloantibodies as in Immune Thrombocytopenia 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 multi-functional hematopoietic receptor agonists of the present invention may be useful in the mobilization of hematopoietic progenitors and stem cells in peripheral blood. Peripheral blood derived progenitors have been shown to be effective in reconstituting patients in the setting of autologous marrow transplantation. Hematopoietic growth factors including G-CSF and GM-CSF have been shown to enhance the number of circulating progenitors and stem cells in the peripheral blood.

This has simplified the procedure for peripheral stem WO 98/46750 PCTIUS98/07511 26 cell collection and dramatically decreased the cost of thp procedure by decreasing the number of pheresis required. The multi-functional hematopoietic receptor agonist may be useful in mobilization of stem cells and further enhance the efficacy of peripheral stem cell transplantation.

The multi-functional hematopoietic receptor agonists of the present invention may also be useful in the ex vivo expansion of hematopoietic progenitors and stem cells. Colony stimulating factors (CSFs), such as hIL-3, have been administered alone, coadministered with other CSFs, or in combination with bone marrow transplants subsequent to high dose chemotherapy to treat the neutropenia and thrombocytopenia which are often the result of such treatment. However the period of severe neutropenia and thrombocytopenia may not be totally eliminated.

The myeloid lineage, which is comprised of monocytes (macrophages), granulocytes (including neutrophils) and megakaryocytes, is critical in preventing infections and bleeding which can be lifethreatening. Neutropenia and thrombocytopenia may also be the result of disease, genetic disorders, drugs, toxins, radiation and many therapeutic treatments such as conventional oncology therapy.

Bone marrow transplants have been used to treat this patient population. However, several problems are associated with the use of bone marrow to reconstitute a compromised hematopoietic system including: 1) the number of stem cells in bone marrow, spleen, or peripheral blood is limited, 2) Graft Versus Host Disease, 3) graft rejection and 4) possible contamination with tumor cells. Stem cells make up a very small percentage of the nucleated cells in the bone marrow, spleen and peripheral blood. It is clear that a dose response exists such WO 98/46750 PCT/US98/07511 27 that a greater number of stem cells will enhance hematopoietic recovery. Therefore, the in vitro expansion of stem cells should enhance hematopoietic recovery and patient survival. Bone marrow from an allogeneic donor has been used to provide bone marrow for transplant. However, Graft Versus Host Disease and graft rejection limit bone marrow transplantation even in recipients with HLA-matched sibling donors.

An alternative to allogeneic bone marrow transplants is autologous bone marrow transplants. In autologous bone marrow transplants, some of the patient's own marrow is harvested prior to myeloablative therapy, e.g. high dose chemotherapy, and is transplanted back into the patient afterwards. Autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection. However, autologous bone marrow transplants still present problems in terms of the limited number of stems cells in the marrow and possible contamination with tumor cells. The limited number of stem cells may be overcome by ex-vivo expansion of the stem cells. In addition, stem cells can be specifically isolated, based on the presence of specific surface antigens such as CD34+ in order to decrease tumor cell contamination of the marrow graft.

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

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

WO 98/46750 PCT/US98/07511 28 5,199,942 describes a method for autologous hematopoietic cell transplantation comprising: (1) obtaining hematopoietic progenitor cells from a patient; ex-vivo expansion of cells with a growth factor selected from the group consisting of IL-3, flt3 ligand, c-kit ligand, GM-CSF, IL-1, GM-CSF/IL-3 chimera 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 mammalian cells derived from peripheral blood.

WO 93/18648 relates to a composition comprising human neutrophil precursor cells with a high content of WO 98/46750 PCT/US98/07511 29 myeloblasts and promyelocytes for treating genetic or acquired neutropenia.

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

WO 94/11493 describes a stem cell population that are CD34+ and small in size, which are isolated using a counterflow elutriation method.

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

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

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

Invest. 86:932-941, 1990).

One aspect of the present invention provides a method for selective ex-vivo expansion of stem cells. The term "stem cell" refers to the totipotent hematopoietic stem cells as well as early precursors and progenitor cells which can be isolated from bone marrow, spleen or peripheral blood. The term "expansion" refers to the differentiation and proliferation of the cells. The present invention provides a method for selective ex-vivo expansion of stem cells, comprising the steps of: separating stem cells from other cells, culturing said WO 98/46750 PCT/US98/07511 separated stem cells with a selective media which contains multi-functional hematopoietic receptor agonist protein(s) and harvesting said stems cells. Stem cells, as well as committed progenitor cells destined to become neutrophils, erythrocytes, platelets, etc. may be distinguished from most other cells by the presence or absence of particular progenitor marker antigens, such as CD34, that are present on the surface of these cells and/or by morphological characteristics. The phenotype for a highly enriched human stem cell fraction is reported as CD34+, Thy-l+ and lin-, but it is to be understood that the present invention is not limited to the expansion of this stem cell population. The CD34+ enriched human stem cell fraction can be separated by a number of reported methods, including affinity columns or beads, magnetic beads or flow cytometry using antibodies directed to surface antigens such as the CD34+. Further, physical separation methods such as counterflow elutriation may be used to enrich hematopoietic progenitors. The CD34+ progenitors are heterogeneous, and may be divided into several subpopulations 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-l). 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, flt3 WO 98/46750 PCT/US98/07511 31 ligand or combinations thereof. The proliferation of the stem cells can be monitored by enumerating the number of stem cells and other cells, by standard techniques hemacytometer, CFU, LTCIC) or by flow cytometry prior and subsequent to incubation.

Several methods for ex-vivo expansion of stem cells have been reported utilizing a number of selection methods and expansion using various colony stimulating factors including c-kit ligand (Brandt et al., Blood 83:1507-1514 [1994], McKenna et al., Blood 86:3413-3420 [1995]), IL-3 (Brandt et al., Blood 83:1507-1514 [1994], Sato et al., Blood 82:3600-3609 [1993]), G-CSF (Sato et al., Blood 82:3600-3609 [1993]), GM-CSF (Sato et al., Blood 82:3600-3609 [1993]), IL-I (Muench et al., Blood 81:3463-3473 [1993]), IL-6 (Sato et al., Blood 82:3600-3609 [1993]), IL-i (Lemoli et al., Exp. Hem. 21:1668-1672 [1993], Sato et al., Blood 82:3600-3609 [1993]), flt3 ligand (McKenna et al., Blood 86:3413 3420 [1995]) and/or combinations thereof (Brandt et al., Blood 83:1507 1514 [1994], Haylock et al., Blood 80:1405- 1412 [1992], Koller et al., Biotechnology 11:358-363 [1993], (Lemoli et al., Exp. Hem. 21:1668-1672 [1993]), McKenna et al., Blood 86:3413-3420 [1995], Muench et al., Blood 81:3463-3473 [1993], Patchen et al., Biotherapy 7:13-26 [1994], Sato et al., Blood 82:3600-3609 [1993], Smith et al., Exp. Hem. 21:870- 877 [1993], Steen et al., Stem Cells 12:214-224 [1994], Tsujino et al., Exp. Hem. 21:1379-1386 [1993]). Among the individual colony stimulating factors, hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609 [1993], Kobayashi et al., Blood 73:1836-1841 [1989]). However, no single factor has been shown to be as effective as the combination of multiple factors. The present WO 98/46750 PCT/S98/07511 32 invention provides methods for ex vivo expansion that utilize multi-functional hematopoietic receptor agonists that are more effective than a single factor alone.

Another aspect of the invention provides methods of sustaining and/or expanding hematopoietic precursor cells which includes inoculating the cells into a culture vessel which contains a culture medium that has been conditioned by exposure to a stromal cell line such as HS-5 (WO 96/02662, Roecklein and Torok-Strob, Blood 85:997-1105, 1995) that has been supplemented with a multi-functional hematopoietic 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 immunodeficiencies (Kay and Woo, Trends Genet. 10:253-257 [1994]), 2) neurological disorders (Friedmann, Trends Genet.

10:210-214 [1994]), 3) cancer (Culver and Blaese, Trends Genet. 10:174-178 [1994]) and 4) infectious WO 98/46750 PCT/UTS98/07511 33 diseases (Gilboa and Smith, Trends Genet. 10:139-144 [1994]).

There are a variety of methods, known to those with skill in the art, for introducing genetic material into a host cell. A number of vectors, both viral and non-viral have been developed for transferring therapeutic genes into primary cells.

Viral based vectors include; 1) replication deficient recombinant retrovirus (Boris-Lawrie and Temin, Curr.

Opin. Genet. Dev. 3:102-109 [1993], Boris-Lawrie and Temin, Annal. New York Acad. Sci. 716:59-71 [1994], Miller, Current Top. Microbiol. Immunol. 158:1-24 [1992]) and replication-deficient recombinant adenovirus (Berkner, BioTechniques 6:616-629 [1988], Berkner, Current Top. Microbiol. Immunol. 158:39-66 [1992], Brody and Crystal, 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 multi-functional hematopoietic receptor agonist proteins that have improved biological activity, including an activity not seen by any single colony stimulation factor.

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 WO 98/46750 PCT/US98/07511 34 as meprobamate, analgesics such as aminopyrine and dipyrone, anti-convulsants such as phenytoin or carbamazepine, antithyroids such as propylthiouracil and methimazole and diuretics. The multi-functional hematopoietic receptor agonists of the present invention may be useful in preventing or treating the bone marrow suppression or hematopoietic deficiencies which often occur in patients treated with these drugs.

Hematopoietic deficiencies may also occur as a result of viral, microbial or parasitic infections and as a result of treatment for renal disease or renal failure, dialysis. The multi-functional hematopoietic receptor agonists of the present invention may be useful in treating such hematopoietic deficiencies.

Various immunodeficiencies, in T and/or B lymphocytes, or immune disorders, rheumatoid arthritis, may also be beneficially affected by treatment with the multi-functional hematopoietic receptor agonists of the present invention.

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

WO 98/46750 PCT/US98/07511 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 multi-functional hematopoietic 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 treatment of hematopoietic deficiency may include administration of a pharmaceutical composition containing the multi-functional hematopoietic receptor agonists to a patient. The multi-functional hematopoietic receptor agonists of the present invention may also be useful for the activation and amplification of hematopoietic precursor cells by treating these cells in vitro with the multi-functional hematopoietic receptor agonist proteins of the present invention prior to injecting the cells into a patient.

The dosage regimen involved in a method for treating the above-described conditions will be determined by the attending physician considering various factors which modify the action of drugs, 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 pg/kg of multi-functional hematopoietic receptor agonist protein per kilogram of body weight.

WO 98/46750 PCT/US98/07511 36 Dosages would be adjusted relative to the activity of a given multi-functional hematopoietic receptor agonist protein 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 multifunctional hematopoietic 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 hematopoietic growth factors or IL-3 variants or growth factors; co-administration with chemotherapeutic drugs and/or radiation; the use of glycosylated multi-functional hematopoietic receptor agonist protein; and various patient-related issues mentioned earlier in this section. As indicated above, the therapeutic method and compositions may also include co-administration or sequential administration other hematopoietic growth factors. A non-exclusive list of other appropriate hematopoietic growth factors, colony stimulating factors (CSFs), cytokines, lymphokines, and interleukins for simultaneous or serial coadministration with the chimeric proteins of the present invention includes GM-CSF, G-CSF, G-CSF Ser 17 c-mpl ligand (also known as TPO or MGDF), M- CSF, erythropoietin (EPO), IL-1, IL-4, IL-2, IL-3, IL-3 variant, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-15, IL-16, LIF, B-cell growth factor, B-cell differentiation factor and eosinophil differentiation factor, stem cell factor (SCF) also known as steel factor or c-kit ligand, SCSF, SDF-1 or combinations thereof. "hIL-3 variant" is defined as a hIL-3 molecule-which has amino acid substitutions and/or portions of hIL-3 deleted as disclosed in WO 94/12638, WO 94/12639 and WO 95/00646, as well as other variants known in the art. The dosage recited WO 98/46750 PCT/US98/07511 37 above would be adjusted to compensate for such additional components in the therapeutic composition.

Progress of the treated patient can be monitored by periodic assessment of the hematological profile, differential cell count and the like.

Determination of activity of the chimeric proteins Biological activity of the multi-functional hematopoietic receptor agonist proteins of the present invention can be determined by DNA synthesis in factor-dependent cell lines or by counting the colony forming units in an in vitro bone marrow assay. The chimeric proteins may be assayed by a number of in vitro and in vivo models known to those skilled in the art. Examples of such assays include put are not limited to: Methylcellulose Assay This assay reflects the ability of colony stimulating factors to stimulate normal bone marrow cells to produce different types of hematopoietic colonies in vitro (Bradley et al., Aust. Exp Biol. Sci. 44:287- 300, 1966), Pluznik et al., J. Cell Comp. Physio 66:319-324, 1965).

Methods Approximately 30 mL of fresh, normal, healthy bone marrow aspirate are obtained from individuals following informed consent. Under sterile conditions samples are diluted 1:5 with a 1X PBS (#14040.059 Life Technologies, Gaithersburg, MD.) solution in a mL conical tube (#25339-50 Corning, Corning MD).

Ficoll (Histopaque 1077 Sigma H-8889) is layered under the diluted sample and centrifuged, 300 x g for min. The mononuclear cell band is removed and WO 98/46750 PCT/US98/07511 38 washed two times in IX PBS and once with 1% BSA PBS (CellPro Co., Bothel, WA). Mononuclear cells are counted and CD34+ cells are selected using the Ceprate LC (CD34) Kit (CellPro Co., Bothel, WA) column. This fractionation is performed since all stem and progenitor cells within the bone marrow display CD34 surface antigen.

Cultures are set up in triplicate with a final volume of 1.0 mL in a 35 X 10 mm petri dish (Nunc#174926).

Culture medium is purchased from Terry Fox Labs.

(HCC-4230 medium (Terry Fox Labs, Vancouver, B.C., Canada) and erythropoietin (Amgen, Thousand Oaks, CA.) is added to the culture media. 3,000-10,000 CD34+ cells are added per dish. Recombinant IL-3, purified from mammalian cells or E. coli, and multifunctional hematopoietic receptor agonist proteins, in conditioned media from transfected mammalian cells or purified from conditioned media from transfected mammalian cells or E. coli, are added to give final concentrations ranging from .001 nM to 10 nM.

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

Control (baseline response) cultures received no colony stimulating factors. Positive control cultures received conditioned media (PHA stimulated human cells: Terry Fox Lab. H2400). Cultures are incubated at 37 0 C, 5% C02 in humidified air.

Hematopoietic colonies which are defined as greater than 50 cells are counted on the day of peak response (days 10-11) using a Nikon inverted phase microscope with a 40x objective combination. Groups of cells containing fewer than 50 cells are referred to as clusters. Alternatively colonies can be identified by WO 98/46750 PCT/US98/07511 39 spreading the colonies on a slide and stained or they can be picked, resuspended and spun onto cytospin slides for staining.

Human Cord Blood Hemopoietic Growth Factor Assays Bone marrow cells are traditionally used for in vitro assays of hematopoietic colony stimulating factor (CSF) activity. However, human bone marrow is not always available, and there is considerable variability between donors. Umbilical cord blood is comparable to bone marrow as a source of hematopoietic stem cells and progenitors (Broxmeyer et al., PNAS USA 89:4109-113, 1992; Mayani et al., Blood 81:3252-3258, 1993). In contrast to bone marrow, cord blood is more readily available on a regular basis. There is also a potential to reduce assay variability by pooling cells obtained fresh from several donors, or to create a bank of cryopreserved cells for this purpose. By modifying the culture conditions, and/or analyzing for lineage specific markers, it is be possible to assay specifically for granulocyte macrophage colonies (CFU-GM), for megakaryocyte CSF activity, or for high proliferative potential colony forming cell (HPP-CFC) activity.

Methods Mononuclear cells (MNC) are isolated from cord blood within 24 hr. of collection, using a standard density gradient (1.077 g/mL Histopaque). Cord blood MNC have been further enriched for stem cells and progenitors by several procedures, including immunomagnetic selection for CD14-, CD34+ cells; panning for SBA-, CD34+ fraction using coated flasks from Applied Immune Science (Santa Clara, CA); and CD34+ selection using a CellPro (Bothell, WA) avidin column. Either freshly isolated or cryopreserved WO 98/46750 PCT/US98/07511 CD34+ cell enriched fractions are used for the assay.

Duplicate cultures for each serial dilution of sample (concentration range from 1 pM to 1204 pM) are prepared with 1x104 cells in 1ml of 0.9% methycellulose containing medium without additional growth factors (Methocult H4230 from Stem Cell Technologies, Vancouver, In some experiments, Methocult H4330 containing erythropoietin (EPO) was used instead of Methocult H4230, or Stem Cell Factor (SCF), 50 ng/mL (Biosource International, Camarillo, CA) was added. After culturing for 7-9 days, colonies containing >30 cells are counted. In order to rule out subjective bias in scoring, assays are scored blind.

AML Proliferation Assay for Bioactive Human Interleukin-3 The factor-dependent cell line AML 193 was obtained from the American Type Culture Collection (ATCC, Rockville, MD). This cell line, established from a patient with acute myelogenous leukemia, is a growth factor dependent cell line which displayed enhanced growth in GM-CSF supplemented medium (Lange, et al., Blood 70: 192, 1987; Valtieri, et al., J. Immunol. 138:4042, 1987). The ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, D., et al., J. Immunol. 139: 348, 1987). A cell line variant was used, AML 193 1.3, which was adapted for long term growth in IL-3 by washing out the growth factors and starving the cytokine dependent AML 193 cells for growth factors for 24 hours. The cells are then replated at 1x10 5 cells/well in a 24 well plate in media containing 100 U/mL IL-3. It took approximately 2 months for the cells to grow rapidly in IL-3. These cells are maintained as AML 193 1.3 WO 98/46750 PCT/US98/07511 41 thereafter by supplementing tissue culture medium (see below) with human IL-3.

AML 193 1.3 cells are washed 6 times in cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, NY) by centrifuging cell suspensions at 250 x g for 10 minutes followed by decantation of the supernatant. Pelleted cells are resuspended in HBSS and the procedure is repeated until six wash cycles are completed. Cells washed six times by this procedure are resuspended in tissue culture medium at a density ranging from 2 x 105 to 5 x 105 viable cells/mL. This medium is prepared by supplementing Iscove's modified Dulbecco's Medium (IMDM, Hazelton, Lenexa, KS) with albumin, transferrin, lipids and 2mercaptoethanol. Bovine albumin (Boehringer- Mannheim, Indianapolis, IN) is added at 500 pg/mL; human transferrin (Boehringer-Mannheim, Indianapolis, IN) is added at 100 pg/mL; soybean lipid (Boehringer- Mannheim, Indianapolis, IN) is added at 50 ug/mL; and 2-mercaptoethanol (Sigma, St. Louis, MO) is added at x 10 5

M.

Serial dilutions of human interleukin-3 or multi-functional chimeric hematopoietic receptor agonist proteins are made in triplicate series in tissue culture medium supplemented as stated above in 96 well Costar 3596 tissue culture plates. Each well contained 50 pl of medium containing interleukin-3 or multi-functional chimeric hematopoietic receptor agonist proteins once serial dilutions are completed.

Control wells contained tissue culture medium alone (negative control). AML 193 1.3 cell suspensions prepared as above are added to each well by pipetting il (2.5 x 104 cells) into each well. Tissue culture plates are incubated at 37 0 C with 5% CO 2 in

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WO 98/46750 PCT/US98/07511 42 humidified air for 3 days. On day 3, 0.5 Ci 3Hthymidine (2 Ci/mM, New England Nuclear, Boston, MA) is added in 50 pl of tissue culture medium. Cultures are incubated at 37 0 C with 5% C0 2 in humidified air for 18-24 hours. Cellular DNA is harvested onto glass filter mats (Pharmacia LKB, Gaithersburg, MD) using a TOMTEC cell harvester (TOMTEC, Orange, CT) which utilized a water wash cycle followed by a ethanol wash cycle. Filter mats are allowed to air dry and then placed into sample bags to which scintillation fluid (Scintiverse II, Fisher Scientific, St. Louis, MO or BetaPlate Scintillation Fluid, Pharmacia LKB, Gaithersburg, MD) is added.

Beta emissions of samples from individual tissue culture wells are counted in a LKB BetaPlate model 1205 scintillation counter (Pharmacia LKB, Gaithersburg, MD) and data is expressed as counts per minute of 3 H-thymidine incorporated into cells from each tissue culture well. Activity of each human interleukin-3 preparation or multi-functional chimeric hematopoietic receptor agonist protein preparation is quantitated by measuring cell proliferation' 3 H-thymidine incorporation) induced by graded concentrations of interleukin-3 or multifunctional chimeric hematopoietic receptor agonist.

Typically, concentration ranges from 0.05 pM 105 pM are quantitated in these assays. Activity is determined by measuring the dose of interleukin-3 or multi-functional chimeric hematopoietic receptor agonist protein which provides 50% of maximal proliferation (EC50 0.5 x (maximum average counts per minute of 3H-thymidine incorporated per well among triplicate cultures of all concentrations of interleukin-3 tested background proliferation WO 98/46750 PCT/US98/07511 43 measured by 3 H-thymidine incorporation observed in triplicate cultures lacking interleukin-3). This ECs value is also equivalent to 1 unit of bioactivity.

Every assay is performed with native interleukin-3 as a reference standard so that relative activity levels could be assigned.

Typically, the multi-functional chimeric hematopoietic receptor agonist proteins were tested in a concentration range of 2000 pM to 0.06 pM titrated in serial 2 fold dilutions.

Activity for each sample was determined by the concentration which gave 50% of the maximal response by fitting a four-parameter logistic model to the data. It was observed that the upper plateau (maximal response) for the sample and the standard with which it was compared did not differ. Therefore relative potency calculation for each sample was determined from EC50 estimations for the sample and the standard as indicated above. AML 193.1.3 cells proliferate in response to hIL-3, hGM-CSF and hG-CSF.

TF1 c-mpl ligand dependent proliferation assay The c-mpl ligand proliferative activity can be assayed using a subclone of the pluripotential human cell line TF1 (Kitamura et al., J. Cell Physiol 140:323-334. [1989]). TF1 cells are maintained in h- IL3 (100 U/mL). To establish a sub-clone responsive to c-mpl ligand, cells are maintained in passage media containing 10% supernatant from BHK cells transfected with the gene expressing the 1-153 form of c-mpl ligand (pMON26448). Most of the cells die, but a subset of cells survive. After dilution cloning, a c-mpl ligand responsive clone is selected, and these cells are split into passage media to a density of 0.3 x 10 cells/mL the day prior to assay WO 98/46750 PCT/US98/07511 44 set-up. Passage media for these cells is the following: RPMI 1640 (Gibco), 10% FBS (Harlan, Lot #91206), 10% c-mpl ligand supernatant from transfected BHK cells, 1 mM sodium pyruvate (Gibco), 2 mM glutamine (Gibco), and 100 ug/mL penicillinstreptomycin (Gibco). The next day, cells are harvested and washed twice in RPMI or IMDM media with a final wash in the ATL, or assay media. ATL medium consists of the following: IMDM (Gibco), 500 ug/mL of bovine serum albumin, 100 ug/mL of human transferrin, ug/mL soybean lipids, 4 x 10-8M betamercaptoethanol and 2 mL of A9909 (Sigma, antibiotic solution) per 1000 mL of ATL. Cells are diluted in assay media to a final density of 0.25 x 106 cells/mL in a 96-well low evaporation plate (Costar) to a final volume of 50 ul. Transient supernatants (conditioned media) from transfected clones are added at a volume of 50 ul as duplicate samples at a final concentration of 50% and diluted three-fold to a final dilution of Triplicate samples of a dose curve of IL-3 variant pMON13288 starting at 1 ng/mL and diluted using three-fold dilutions to 0.0014ng/mL is included as a positive control. Plates are incubated at 5% CO 2 and 370 C. At day six of culture, the plate is pulsed with 0.5 Ci of 3H/well (NEN) in a volume of 20 ul/well and allowed to incubate at 5% CO 2 and 370 C for four hours. The plate is harvested and counted on a Betaplate counter.

MUTZ-2 Cell Proliferation Assay WO 98/46750 PCT/US98/07511 A cell line such as MUTZ-2, which is a human myeloid leukemia cell line (German Collection of Microorganisms and Cell Cultures, DSM ACC 271), can be used to determine the cell proliferative activity of flt3 receptor agonists. MUTZ-2 cultures are maintained with recombinant native flt3 ligand 100ng/mL) in the growth medium. Eighteen hours prior to assay set-up, MUTZ-2 cells are washed in IMDM medium (Gibco) three times and are resuspended in IMDM medium alone at a concentration of 0.5-0.7 x 10E6 cells/mL and incubated at 37 0 C and 5%C0 2 to starve the cells of flt3 ligand. The day of the assay, standards and flt3 receptor agonists are diluted to two fold above desired final concentration in assay media in sterile tissue culture treated 96 well plates. Flt3 receptor agonists and standards are tested in triplicate. 501l of assay media is loaded into all wells except row A. 75pl of the flt3 receptor agonists or standards are added to row A and 254l taken from that row and serial dilutions (1:3) performed on the rest of the plate (rows B through Row H remains as a media only control. The starved MUTZ-2 cells are washed two times in IMDM medium and resuspended in 50pl assay media. 50l of cells are added to each well resulting in a final concentration of 0.25 x 10E6cells/mL. Assay plates containing cells are incubated at 37 0 C and 5%C0 2 for 44hrs. Each well is then pulsed with 14Ci/well of tritiated thymidine in a volume of 20pl for four hours. Plates are then harvested and counted.

Other in vitro cell based proliferation assays WO 98/46750 PCTIUS98/07511 46 Other in vitro cell based assays, known to those skilled in the art, may also be useful to determine the activity of the multi-functional chimeric hematopoietic receptor agonists depending on the factors that comprise the molecule in a similar manner as described in the AML 193.1.3 cell proliferation assay. The following are examples of other useful assays.

TF1 proliferation assay: TF1 is a pluripotential human cell line (Kitamura et al., J. Cell Physiol 140:323-334. [1989]) that responds to hIL-3.

32D proliferation assay: 32D is a murine IL-3 dependent cell line which does not respond to human IL-3 but does respond to human G-CSF which is not species restricted.

Baf/3 proliferation assay: Baf/3 is a murine IL-3 dependent cell line which does not respond to human IL-3, human flt3 ligand or human c-mpl ligand but does respond to human G-CSF which is not species restricted.

T1165 proliferation assay: T1165 cells are a IL-6 dependent murine cell line (Nordan et al., 1986) which respond to IL-6 and IL-11.

Human Plasma Clot meg-CSF Assay: Used to assay megakaryocyte colony formation activity (Mazur et al., 1981).

Transfected cell lines: Cell lines such as the murine Baf/3 cell line can be transfected with a hematopoietic growth factor WO 98/46750 PCT/US98/07511 47 receptor, such as the human G-CSF receptor or human c-mpl receptor, which the cell line does not 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 Baf/3 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.

In a similar manner the human G-CSF receptor can be transfected into the Baf/3 cell line and used to determine the bioactivity of the multi-functional chimeric hematopoietic receptor agonists.

Analysis of c-mpl ligand proliferative activity Methods 1. Bone marrow proliferation assay a. CD34+ Cell Purification: Bone marrow aspirates (15-20 mL) were obtained from normal allogeneic marrow donors after informed consent. Cells were diluted 1:3 in phosphate buffered saline (PBS, Gibco-BRL), 30 mL were layered over 15 mL Histopaque-1077 (Sigma) and centrifuged for 30 minutes at 300 RCF. The mononuclear interface layer was collected and washed in PBS. CD34+ cells were enriched from the mononuclear cell preparation using an affinity column per manufacturers WO 98/46750 PCT/US98/07511 48 instructions (CellPro, Inc., Bothell WA). After enrichment, the purity of CD34+ cells was 70% on average as determined by using flow cytometric analysis using anti-CD34 monoclonal antibody conjugated to fluorescein and anti-CD38 conjugated to phycoerythrin (Becton Dickinson, San Jose CA).

Cells were resuspended at 40,000 cells/mL in X- Vivo 10 media (Bio-Whittaker, Walkersville, MD) and 1 mL was plated in 12-well tissue culture plates (Costar). The growth factor rhIL-3 was added at 100 ng/mL (pMON5873) was added to some wells. hIL3 variants were used at 10 ng/mL to 100 ng/mL.

Conditioned media from BHK cells transfected with plasmid encoding c-mpl ligand or multi-functional chimeric hematopoietic receptor agonists were tested by addition of 100 l of supernatant added to 1 mL cultures (approximately a 10% dilution). Cells were incubated at 37 0 C for 8-14 days at 5% CO 2 in a 37 0

C

humidified incubator.

b. Cell Harvest and Analysis: At the end of the culture period a total cell count was obtained for each condition. For fluorescence analysis and ploidy determination cells were washed in megakaryocyte buffer (MK buffer, 13.6 mM sodium citrate, 1 mM theophylline, 2.2 um PGE1, 11 mM glucose, 3% w/v BSA, in PBS, pH (Tomer et al., Blood 70: 1735-1742, 1987) resuspended in 500 pl of MK buffer containing anti-CD41a FITC antibody (1:200, AMAC, Westbrook, ME) and washed in MK buffer.

For DNA analysis cells were permeablized in MK buffer containing 0.5% Tween 20 (Fisher, Fair Lawn NJ)for min. on ice followed by fixation in 0.5% Tween-20 and 1% paraformaldehyde (Fisher Chemical) for 30 minutes followed by incubation in propidium iodide WO 98/46750 PCT/US98/07511 49 (Calbiochem La Jolla Ca) (50 pg/mL) with RNAase (400 U/mL) in 55% v/v MK buffer (200mOsm) for 1-2 hours on ice. Cells were analyzed on a FACScan or Vantage flow cytometer (Becton Dickinson, San Jose, CA). Green fluorescence (CD41a-FITC) was collected along with linear and log signals for red fluorescence (PI) to determine DNA ploidy. All cells were collected to determine the percent of cells that were CD41+. Data analysis was performed using software by LYSIS (Becton Dickinson, San Jose, CA).

Percent of cells expressing the CD41 antigen was obtained from flow cytometry analysis(Percent).

Absolute (Abs) number of CD41+ cells/mL was calculated by: (Abs)=(Cell Count)*(Percent)/100.

2. Megakaryocyte fibrin clot assay.

CD34+ enriched population were isolated as described above. Cells were suspended at 25,000 cells/mL with or without cytokine(s) in a media consisting of a base Iscoves IMDM media supplemented with 0.3% BSA, 0.4mg/mL apo-transferrin, 6.67pM FeC12, CaC1 2 25pg/mL L-asparagine, 500g/mL e-amino-ncaproic acid and penicillin/streptomycin. Prior to plating into 35mm plates, thrombin was added (0.25 Units/mL) to initiate clot formation. Cells were incubated at 37 0 C for 13 days at 5% CO 2 in a 37 0

C

humidified incubator.

At the end of the culture period plates were fixed with methanol:acetone air dried and stored at -200C until staining. A peroxidase immunocytochemistry staining procedure was used (Zymed, Histostain-SP. San Francisco, CA) using a WO 98/46750 PCT/US98/07511 cocktail of primary monoclonal antibodies consisting of anti-CD41a, CD42 and CD61. Colonies were counted after staining and classified as negative, CFU-MK (small colonies, 1-2 foci and less that approx. cells), BFU-MK (large, multi-foci colonies with cells) or mixed colonies (mixture of both positive and negative cells.

EXAMPLES 1 2 Construction of an expression vectors, pMON32364 and pMON32377, comprising a DNA sequence encoding a multi-functional receptor agonist comprising an IL-3 (15-125) variant joined via the IgG2b linker to a flt-3 (1-134) ligand and an IL-3 (15-125) variant joined via the IgG2b linker to a flt-3 (1-139) ligand, respectively. Plasmids, pMON32364 and pMON32377, were assembled by cloning gel-purified NcoI/HindIII DNA fragments from pMON30237 SEQ ID NO:53 and pMON30238 SEQ ID NO:54, containing the flt-3 (1-134) ligand and flt-3 (1- 139) into vector, pMON30311 (derivative of pMON13058 W095/21254), DNA digested with AflIII/HindIII and SAP-treated (pMON30311 is a BHK-specific vector containing NcoI-IL-3 receptor agonist-IgG2B-AflIII-HindIII as an insert) using standard ligation conditions. The ligation mixtures were used to transform competent cells (Gibco BRL cat #18265-017) following the manufacturer's recommended protocol, and vector WO 98/46750 PCT/US98/07511 51 DNA was isolated from ampicillin-resistant colonies. The DNA sequence of resulting genes (SEQ ID NO:21 and SEQ ID NO:22 respectively) were determined by automated fluorescent DNA sequencing on an ABI 373/377 DNA sequencer (Perkin Elmer ABI) using Sequencher (Gene Codes) software. The resulting vectors, pMON32364 and pMON32377, encodes the proteins of SEQ ID NO:42 and SEQ ID NO:43 respectively.

EXAMPLES 3 4 Construction of an expression vectors, pMON30247 and pMON30246, comprising a DNA sequence encoding a multi-functional receptor agonist comprising an IL-3 (15-125) variant joined via the IgG2b linker to a flt-3 (1-134) ligand and an IL-3 (15-125) variant joined via the GlySer linker to a flt-3 (1-134) ligand, respectively. Plasmids, pMON30246 and pMON30247, were constructed by cloning the gel purified NcoI/AflIII restriction fragment from pMON30244 (GlySer linker) SEQ ID and pMON30245 (IgG2B linker) SEQ ID NO:66 respectively, into vector, pMON30237, digested with NcoI (which contains hFlt3L 1-134) as described in Examples 1 2. The DNA sequence of resulting genes, SEQ ID NO:13 and SEQ ID NO:14, WO 98/46750 PCT/US98/07511 52 encodes the proteins of SEQ ID NO:42 and SEQ ID NO:43, respectively.

EXAMPLES 5 6 Construction of an expression vectors, pMON30249 and pMON30248, comprising a DNA sequence encoding a multi-functional receptor agonist comprising an IL-3 (15-125) variant joined via the IgG2b linker to a flt-3 (1-139) ligand and an IL-3 (15-125) variant joined via the GlySer linker to a flt-3 (1-139) ligand, respectively. Plasmids, pMON30248 and pMON30249, were constructed by cloning the gel purified NcoI/AflIII restriction fragment from pMON30244 (GlySer linker) and pMON30245 (IgG2B linker) respectively, into vector, pMON30238, digested with NcoI (which contains hFlt3L 1-139) as described in Examples 1 2. The DNA sequence of resulting genes, SEQ ID NO:15 and SEQ ID NO:16, encodes the proteins of SEQ ID NO:36 and SEQ ID NO:37, respectively.

EXAMPLES 7 8 Construction of an expression vectors, pMON32392 and pMON32393, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-134) ligand joined via the WO 98/46750 PCT/US98/07511 53 IgG2b linker to an IL-3 (15-125) variant and a flt-3 (1-139) ligand joined via the IgG2b linker to an IL-3 (15-125) variant, respectively.

Plasmids, pMON32392 and pMON32393, were constructed by Polymerase Chain Reaction (PCR) methods. Plasmid, pMON30237 and pMON30238, DNA was used as the template in the PCR reaction with primer pairs N-term SEQ ID NO:29/134rev SEQ ID and N-term SEQ ID NO:29/139rev SEQ ID NO:31, respectively, to introduce an in-frame SnaBI restriction site at the C-terminus. Standard PCR reaction mixtures were set up using an Invitrogen PCR Optimizer kit (Invitrogen). Amplification cycle conditions were as follows: seven cycles of 94 0 C, one minute, 650C, two minutes, and 72 0 C 2 1/2 minutes; followed by ten cycles of 94°C, one minute, 70 0 C, two minutes, and 72°C 2 1/2 minutes. The product of the PCR reactions were purified using the Wizard PCR Purification kit (Promega), and eluted in 50 .l dH 2 0. 20 p1 of each purified PCR product were digested in 50 1l reaction mixture volumes with 10U each of NcoI and SnaBI for 90 minutes at 37 0 C. One pg of vector, pMON26431 (derivative of pMON13061 W095/21254), was digested with 7.5U each of NcoI and SnaBI in a .l reaction volume for 90 minutes at 37 0

C,

followed by the addition of 1U shrimp alkaline phosphatase. The reaction was incubated an WO 98/46750 PCT/US98/07511 54 additional 10 minutes at 37 0 C, and both inserts and vector were gel purified as described previously. Ligation times and temperatures were modified to include incubation at 16 0 C for 3 hour, followed by 2 hour at ambient temperature.

Transformations and DNA sequence confirmation were done as described in Examples 1 2. The DNA sequence of resulting genes, SEQ ID NO:23 and SEQ ID NO:24, encodes the proteins of SEQ ID NO:44 and SEQ ID NO:45, respectively.

EXAMPLE 9 Construction of an expression vector, pMON30328, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-134) ligand joined via the IgG2b linker to a G-CSF receptor agonist. Plasmid, pMON30328, was constructed by subcloning the gel-purified NcoI/HindIII restriction fragment from pMON30237 into plasmid, pMON30309 (derivative of pMON13149 W095/21254), digested with AflIII/HindIII (contains G-CSF/IgG2b-AflIII/HindIII) as described in Examples 1 2. The DNA sequence of resulting gene, SEQ ID NO:50, encodes the protein of SEQ ID EXAMPLE WO 98/46750 PCT/US98/07511 Construction of an expression vector, pMON30329, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a G- CSF receptor agonist joined via the IgG2b linker to a flt3 (1-139) ligand. Plasmid, pMON30329, was constructed by subcloning the gel-purified NcoI/HindIII restriction fragment from pMON30238 into plasmid pMON30309 digested with AflIII/HindIII (which contains G-CSF/IgG2b- AflIII/HindIII) as described in Examples 1 2.

The DNA sequence of resulting gene, SEQ ID NO:17, encodes the protein of SEQ ID NO:38.

EXAMPLE 11 Construction of an expression vector, pMON32175, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-139) ligand joined via the IgG2b linker to a G-CSF receptor agonist. Plasmid, pMON32175, was constructed by subcloning the gel-purified NcoI/SnaBI restriction fragment from pMON32393 into pMON26430 (derivative of pMON13060 W095/21254) digested with NcoI/SnaBI as described in Examples 1 2. The DNA sequence of resulting gene, SEQ ID NO:19, encodes the protein of SEQ ID WO 98/46750 PCTfS98/07511 56 EXAMPLE 12 Construction of an expression vector, pMON32191, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-139) ligand joined via the IgG2b linker to a G-CSF receptor agonist. pMON32191 was assembled by subcloning the gel-purified NcoI/SnaBI restriction fragment from pMON32393 SEQ ID NO:58 into plasmid pMON31123 digested with NcoI/SnaBI (which contains the GlySer/G-CSF moiety) as described in Examples 1 2. The DNA sequence of resulting gene, SEQ ID NO:20, encodes the protein of SEQ ID NO:41.

EXAMPLE 13 Construction of an expression vector, pMON35767, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-139) ligand joined via the IgG2b linker to a G-CSF receptor agonist. Plasmid, pMON35767, was constructed by subcloning the gel-purified NcoI/HindIII restriction fragment from pMON32191 SEQ ID NO:20 into the BHK expression vector pMON3934, which is a derivative of pMON3359.

pMON3359 is a pUC18-based vector containing a WO 98/46750 PCT/US98/07511 57 mammalian expression cassette. The cassette includes a herpes simplex viral promoter IE110 800 to +120) followed by a modified human IL-3 signal peptide sequence and an SV40 late polyadenylation (poly-A) signal which has been subcloned into the pUC18 polylinker (See Hippenmeyer et al., Bio/Technology, 1993, pp.1037- 1041). The DNA sequence of resulting gene, SEQ ID encodes the protein of SEQ ID NO:41.

EXAMPLE 14 Construction of an expression vector, pMON32173, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-139) ligand joined via the IgG2b linker to a flt3 (1-139) ligand. Plasmid, pMON32173, was constructed by subcloning the gel-purified -130bp NcoI/SacI restriction fragment from pMON32342 SEQ ID NO:52 and the -290bp SacI/SnaBI restriction fragment from pMON32393 into plasmid pMON30329 digested with NcoI/SnaBI as described in Examples 1 2. The DNA sequence of resulting gene, SEQ ID NO:18, encodes the protein of SEQ ID NO:39.

EXAMPLE Construction of an expression vector, pMON45419, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a c-mpl (1-153) ligand joined via the IgG2b linker to a flt3 (1-139) ligand.

Plasmid, pMON45419, was constructed by subcloning the WO 98/46750 PCT/US98/07511 58 NcoI/SnaBI restriction fragment from pMON26474 (derivative of pMON26472 W095/21254) into plasmid, pMON32173 SEQ ID NO:56, digested with NcoI/SnaBI as described in Examples 1 2. The DNA sequence of resulting gene, SEQ ID NO:25, encodes the protein of SEQ ID NO:46.

EXAMPLE 16 Construction of an expression vector, pMON45420, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-139) ligand joined via the IgG2b linker to a c-mpl (1-153) ligand. Plasmid, pMON45420 (derivative of pMON26471 W095/21254) was assembled by subcloning the NcoI/SnaBI restriction fragment from pMON32191 into plasmid, pMON26473, digested with NcoI/SnaBI as described in Examples 1 2. The DNA sequence of resulting gene, SEQ ID NO:26, encodes the protein of SEQ ID NO:47.

EXAMPLE 17 Construction of plasmid pMON46408 which encodes a multi-functional receptor agonist comprising EPO joined via a Gly Ser linker to a flt3 (1-139) ligand Plasmid pMON46408 was constructed in a two step cloning procedure. First, an intermediate plasmid, pMON46406, was constructed. This plasmid encodes the human EPO sequence joined to a GlySer linker sequence containing the restriction enzyme sites AflIII and HindIII. The following three DNA fragments were ligated together to form plasmid pMON46406: WO 98/46750 PCTUS98/07511 59 1. A 480 bp NcoI-StuI fragment encoding EPO except for the terminal 6 amino acids 2. The annealed oligonucleotides epostu-xma.seq SEQ ID NO: 32 and epostu-xma.rev SEQ ID NO:33 which comprises a StuI-XmaI fragment encoding the terminal 6 amino acids of EPO and a portion of the GlySer polypeptide linker sequence 3. The 3,052 bp NcoI-XmaI vector fragment of plasmid pMON13180 The ligation mixture was used to transform competent MON105 cells and transformants were selected on LB Amp plates. Colonies were picked and analyzed by DNA sequencing analysis to identify a correct clone. A correct clone was assigned pMON46406.

In order to construct pMON46408, plasmid pMON46406 was digested with AflIII and HindIII and the vector portion was purified. This was ligated with the 423 bp NcoI-HindIII fragment of plasmid pMON32342 SEQ ID NO:52, which encodes the flt-3 (1-139) ligand. The ligated ligation mixture was used to transform competent MON105 cells and transformants were selected on LB Amp plates.

Colonies were picked and analyzed by DNA sequencing analysis to identify a correct clone.

A correct clone was assigned pMON46408. The DNA sequence of resulting gene, SEQ ID NO:28, encodes the protein of SEQ ID NO:49.

EXAMPLE 18 Determination of bioactivity of selected chimera proteins WO 98/46750 PCT/US98/07511 Selected chimeras of the present invention were assayed in a Baf3 cell line transfected with the flt3/flk2 receptor (Baf3/flt3) to determine flt3 ligand bioactivity.

Table 1 Baf3/flt3 assay pMON30249 Comparable to native flt3 ligand alone pMON32173 Comparable to native flt3 ligand alone pMON32392 Comparable to native flt3 ligand alone pMON32393 Comparable to native flt3 ligand alone pMON32364 Comparable to native flt3 ligand alone pMON32377 Comparable to native flt3 ligand alone 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 WO 95/00646, WO 94/12639, WO 94/12638, WO 95/20976, WO 95/21197, WO 95/20977, WO 95/21254 and US 08/383,035 which are hereby incorporated by reference in their entirety.

Further details known to those skilled in the art may be found in T. Maniatis, et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, 1982) and references cited therein, incorporated herein by reference; and in J. Sambrook, et al., Molecular Cloning, A Laboratory Manual, 2nd 17/06/2882 11:16 61-3-9696-5881 E.F. WELLINGTON CO PAGE 09 61 edition, Cold Spring Harbor Laboratory, 1989) and references cited therein, incorporated herein by reference. Protein purification methods know to those skilled in the art are described in detail in Methods in Enzymology, Volume 182 'Guide to Protein Purification' edited by Murray Deutscher, Academic Press, San Diego, CA (1990).

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

Various other examples will be apparent to the person skilled in the art after reading the present disclosure without departing from the spirit and 9. scope of the invention. It is intended that all such 15 other examples be included within the scope of the appended claims.

With reference to the use of the word(s) "comprise" or "comprises" or "comprising" in the foregoing description and/or in the following claims, unless the context requires otherwise, those words are used on the basis and clear understanding that they are to be interpreted inclusively, rather than exclusively, and that each of those words is to be so interpreted in construing 9 the foregoing description and/or the following claims.

9*9999 WO 98/46750 PCT/US98/07511 SEQUENCE LISTING GENERAL INFORMATION APPLICANT: G. D. Searle Corporate Patent Department (ii) TITLE OF THE INVENTION: Flt3 Ligand Chimeric Protein (iii) NUMBER OF SEQUENCES: (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: G. D. Searle Corporate Patent Department STREET: P.O. Box 55110 CITY: Chicago STATE: IL COUNTRY: USA ZIP: 60680 COMPUTER READABLE FORM: MEDIUM TYPE: Diskette COMPUTER: IBM Compatible OPERATING SYSTEM: DOS SOFTWARE: FastSEQ for Windows Version (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: FILING DATE: 10-APR-1998

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: 08/837,026 FILING DATE: 11-APR-1997 (viii) ATTORNEY/AGENT INFORMATION: NAME: Bennett, Dennis A REGISTRATION NUMBER: 34,547 REFERENCE/DOCKET NUMBER: C-3018/1/PCT (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: 314-737-6986 TELEFAX: 314-737-6972

TELEX:

INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 4 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 98/46750 PCT/US98/07511 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Gly Gly Gly Ser 1 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Gly Gly Gly Gly Ser 1 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 6 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: Gly Gly Gly Gly Gly Ser 1 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 2 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Gly Ser 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 3 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/46750 PCT/US98/07511 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Gly Ser 1 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 36 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly 1 5 10 Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser 25 Gly Gly Gly Ser INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 24 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Ile Ser Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro 1 5 10 Ser Lys Glu Ser His Lys Ser Pro INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 28 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Ile Glu Gly Arg Ile Ser Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn 1 5 10 WO 98/46750 PCT/US98/07511 Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 349 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro Ile Ser Ser Asp Phe 1 5 10 Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro 25 Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu 40 Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val 55 Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu Ile 70 75 His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg 90 Phe Val Gin Thr Asn Ile Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin 100 105 110 Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gin Asn Phe Ser Arg Cys 115 120 125 Leu Glu Leu Gin Cys Gin Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly 130 135 140 Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn 145 150 155 160 Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr 165 170 175 Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys Ser 180 185 190 Leu Glu Gin Val Arg Lys Ile Gin Gly Asp Gly Ala Ala Leu Gin Glu 195 200 205 Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu 210 215 220 Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro 225 230 235 240 Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser Gly 245 250 255 Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly Ile Ser Pro 260 265 270 Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp Phe 275 280 285 Ala Thr Thr Ile Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala 290 295 300 Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin 305 310 315 320 Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu 325 330 335 Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro WO 98/46750 WO 9846750PCJ'/US98/0751 1 INFORMATION FOR SEQ ID NO:1O: SEQUENCE CHARACTERISTICS: LENGTH: 1047 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCCACCCAGG

CGTGAGCTGT

120

GACGAGGAGC

180

CTCAAGACTG

240

CACTTTGTCA

300

AACATCTCCC

360

ACTCGCCAGA

420

TACGTAGAGG

480

CCGTCTCCTC

540

GCCAGCTCCC

600

GGCGATGGCG

660

GAGCTGGTGC

720

AGCCAGGCCC

780

CAGGGGCTCC

840

CTGCAGCTGG

900

ATGGCCCCTG

960

CGCCGGGCAG

1020

CGCGTTCTAC

1047 SEQUENCE DESCRIPTION: SEQ ID NO:l0: ACTGCTCCTT TCAACACAGC CCCATCTCCT CCGACTTCGC

CTGACTACCT

TCTGCGGGGG

TCGCTGGGTC

CCAAATGTGC

GCCTCCTGCA

ACTTCTCCCG

GCGGTGGAGG

CGTCTAAAGA

TGCCCCAGAG

CAGCGCTCCA

TGCTCGGACA

TGCAGCTGGC

TGCAGGCCCT

ACGTCGCCGA

CCCTGCAGCC

GAGGGGTCCT

GCCACCTTGC

GCTTCAAGAT

CCTCTGGCGG

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

CTCCCCGGGT

ATCTCATAAA

CTTCCTGCTC

GGAGAAGCTG

CTCTCTGGGC

AGGCTGCTTG

GGAAGGGATA

CTTTGCCACC

CACCCAGGGT

GGTTGCTAGC

GCAGCCC

TACCCAGTCA

CTGGTCCTGG

GGCTTGCTGG

CCCCCCAGCT

GAGCAGCTGG

CTGCAGTGTC

GAACCGTCTG

TCTCCAAACA

AAGTCTTTAG

TGTGCCACCT

ATCCCCTGGG

AGCCAACTCC

TCCCCCGAGT

ACCATCTGGC

GCCATGCCGG

CATCTGCAGA

CCGTGGCCTC

CACAGCGCTG

AGCGCGTGAA

GTCTTCGCTT

TGGCGCTGA

AGCCCGACTC

GTC!CAATCTC

TGGCTACACC

AGCAAGTGAG

ACAAGCTGTG

CTCCCCTGAG

ATAGCGGCCT

TGGGTCCCAC

AGCAGATGGA

CCTTCGCCTC

GCTTCCTGGA

TGTCAAAATC

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCATGGATC

CTCAACCCTG

TACTATCAAC

ATTGGGCCCT

AAAGATCCAG

CCACCCCGAG

CTCCTGCCCC

TTTC!CTCTAC

CTTGGACACA

AGAACTGGGA

TGCTTTCCAG

GGTGTCGTAC

INFORMATION FOR SEQ ID NO:l1: SEQUENCE CHARACTERISTICS: LENGTH: 349 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear WO 98/46750 (ii) MOLECULE (xi) SEQUENCE 67 TYPE: protein DESCRIPTION: SEQ ID NO:1l: PCTIUS98/0751

I

Ala Thr Pro Leu Gly Pro Ala Ser Ser Len 1 Lys Gin Val Cys Ser Ser Asp Pro Phe 145 Phe Val Thr Met Phe 225 Pro Len Val Ile Arg 305 Gin Cys Ser Gin Len Pro Gly Pro Phe Ala 130 Gin Len Gin Ile Ala 210 Ala V~al Trp Ala His 290 Phe Leu Leu Leu Lys Leu Ser Len Gin Ala 115 Leu Arg Glu Gly Asn 195 Thr Val Thr Arg Gly 275 Phe Val Val Glu Gin Leu Gly Gin Phe Leu 100 Thr Gin Arg Val Gly 180 Pro Gin Lys Val Leu 260 Ser Val Gin Ala Leu 340 5 Gin Cys His Ala Leu Giy Thr Pro Ala Ser 165 Gly Ser Asp Ile Ala 245 Val Lys Thr Thr Leu 325 Gin Val Ala Ser Leu 70 Tyr Pro Ile Thr Gly 150 Tyr Gly Pro Cys Arg 230 Ser Leu Met Lys Asn 310 Lys Cys Arg Thr Len 55 Gin Gin Thr Trp Gin 135 Gly Arg Ser Pro Ser 215 Gin Asn Ala Gin Cys 295 Ile Pro Gin Lys Tyr Gly Leu Gly Leu Gin 120 Gly Val Val Pro Ser 200 Phe Len Leu Gin Gly 280 Ala Ser Trp, Pro Ile Lys Ile Ala Leu Asp 105 Gin Ala Len Leu Gly 185 Lys Gin Ser Gin Arg 265 Leu Phe Arg Ile Asp 10 Gin Len Pro Gly Len 90 Thr Met Met Val Arg 170 Gin G in His Asp Asp 250 Trp Len Gin Len Thr 330 Ser Pro Giy Cys Trp, Cys 75 Gin Len Gin Pro Ala 155 His Pro Ser Ser Tyr 235 Gin Met Gin Pro Len 315 Arg Ser Gin Asp His Ala Leu Ala Gin Glu Al a 140 Ser Len Ser His Pro 220 Len Gin Gin Arg Pro 300 Gin Gin Thr Leu Ala Giu Ser Len Giy Val Met Ser Gin Pro 175 Ile Pro Ser Asp Gly 255 Lys Thr Cys Ser Ser 335 Len Leu Leu Ser His Ile Ala Ala Ala Ser 160 Tyr Ser Asn Asp Tyr 240 Gly Thr Gin Len Gin 320 Arg INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 1047 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 98/46750 (xi)

GCTACACCAT

CAAGTGAGAA

120

AAGCTGTGCC

180

CCCCTGAGCT

240

AGCGGCCTTT

300

GGTCCCACCT

360

CAGATGGAAG

420

TTCGCCTCTG

480

TTCCTGGAGG

540

GGAGGCTCCC

600

AAAGAATCTC

660

ATCTCCTCCG

720

CCAGTCACCG

780

GTCCTGGCAC

840

TTGCTGGAGC

900

CCCAGCTGTC

960

CAGCTGGTGG

1020

CAGTGTCAGC

1047 PCT/US98/0751 I SEQUENCE DESCRIPTION: SEQ ID NO:12: TGGGCCCTGC CAGCTCCCTG

'AGATCCAGGG

ACCCCGAGGA

CCTGCCCCAG

TCCTCTACCA

TGGACACACT

AACTGGGAAT

CTTTCCAGCG

TGTCGTACCG

CGGGTGAACC

ATAAATCTCC

ACTTCGCTGT

TGGCCTCCAA

AGCGCTGGAT

GCGTGAACAC

TTCGCTTCGT

CGCTGAAGCC

CCGACTCCTC

CGATGGCGCA

GCTGGTGCTG

CCAGGCCCTG

GGGGCTCCTG

GCAGCTGGAC

GGCCCCTGCC

CCGGGCAGGA

CGTTCTACGC

GTCTGGTCCA

AAACATGGCT

CAAAATCCGT

CCTGCAGGAC

GGAGCGGCTC

GGAGATACAC

CCAGACCAAC

CTGGATCACT

AACCCTG

CCCCAGAGCT

GCGCTCCAGG

CTCGGACACT

CAGCTGGCAG

CAGGCCCTGG

GTCGC!CGACT

CTGCAGCCCA

GGGGTCCTGG

CACCTTGCGC

ATCTCTACTA

ACCCAGGACT

GAGCTGTCTG

GAGGAGCTCT

AAGACTGTCG

TCCTGCTCAA

AGAAGCTGTG

CTCTGGGCAT

GCTGCTTGAG

AAGGGATATC

TTGCCACCAC

CCCAGGGTGC

TTGCTAGCCA

AGCCCTACGT

TCAACCCGTC

GCTCCTTCCA

ACTACCTGCT

GCGGGGGCCT

CTGGGTCCAA

GTCTTTAGAG

TGCCACCTAC

CCCCTGGGCT

CCAACTCCAT

CCCCGAGTTG

CATCTGGCAG

CATGCCGGCC

TCTGCAGAGC

AGAGGGCGGT

TCCTCCGTCT

ACACAGCCCC

TCAAGATTAC

CTGGCGGCTG

GATGCAAGGC

TCAGCCCCCC

GACCTCCGAG

CCTGGAGCTG

TTTGTCACCA AATGTGCCTT ATCTCCCGCC TCCTGCAGGA CGCCAGAACT TCTCCCGGTG INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 798 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT 120 CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT 180 ATTGAGGCA TTCTTCGTA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT 240 WO 98/46750

CGACATCCAA

300

TATCTGGTTA

360

CCGGGTGGTG

420

AGCCCCATCT

480

GATTACCCAG

540

CGGCTGGTCC

600

CAAGGCTTGC

660

CCCCCCCCCA

720

TCCGAGCAGC

780

GAGCTGCAGT

798

TCATCATCAA

CCCTTGAGCA

'GTTCTGGCGG

CCTCCGACTT

TCACCGTGGC

TGGCACAGCG

TGGAGCGCGT

GCTGTCTTCG

TGGTGGCGCT

GTCAGCCC

GGCAGGTGAC

AGCGCAGGAA

CGGCTCCAAC

CGCTGTCAAA

CTCCAACCTG

CTGGATGGAG

GAACACGGAG

CTTCGTCCAG

GAAGCCCTGG

TGGCAAGAAT

CAACAGTACG

ATGGCCACCd

ATCCGTGAGC

CAGGACGAGG

CGGCTCAAGA

ATACACTTTG

ACCAACATCT

ATCACTCGCC

TCCGGGAAAA

TAGAGGGCGG

AGGACTGCTC

TGTCTGACTA

AGCTCTGCGG

CTGTCGCTGG

TCACCAAATG

CCCGCCTCCT

AGAACTTCTC

PCT/US98/0751 1

ACTGACGTTC

TGGAGGCTCC

CTTCCAACAC

CCTGCTTCAA

GGCGCTCTGG

GTCCAAGATG

TGCCTTTCAG

GCAGGAGACC

CCGGTGCCTG

INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 843 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT

TTGCTGGACC

120

CGACTTCCAA

180

ATTGAGGCAA

240

CGACATCCAA

300

TATCTGGTTA

360

CCGGGTGAAC

420

CATAAATCTC

480

GACTTCGCTG

540

GTGGCCTCCA

600

CAGCGCTGGA

660

CGCGTGAACA

720

CTTCGCTTCG

780

CGAACAACCT

ACCTGGAGAG

TTCTTCGTAA

TCATCATCAA

CCCTTGAGCA

CGTCTGGTCC

CAAACATGGC

TCAAAATCCG

ACCTGCAGGA

TGGAGCGGCT

CGGAGATACA

CAATGACGAA GACGTCTCTA

CTTCGTAAGG

TCTCCAACCA

GGCAGGTGAC

AGCGCAGGAA

AATCTCTACT

CACCCAGGAC

TGAGCTGTCT

CGAGGAGCTC

CAAGACTGTC

CTTTGTCACC

GCTGTCAAGA

TGTCTGCCCT

TGGCAAGAAT

CAACAGTACG

ATCAACCCGT

TGCTCCTTCC

GACTACCTGC

TGCGGGGCGC

GCTGGGTCCA

AAATGTGCCT

TCCTGATGGA

ACTTAGAAAA

CTGCCACGGC

TCCGGGAAAA.

TAGAGGGCGG

CTCCTCCGTC

AKCACAGCCC

TTCAAGATTA

TCTGGCGGCT

AGATGCAAGG

TTCAGCCCCC

CCGAAACCTT

TGCATCAGGT

CGCACCCTCT

ACTGACGTTC

TGGAGGCTCC

TAAAGAATCT

CATCTCCTCC

CCCAGTCACC

GGTCCTGGCA

CTTGCTGGAG

CCCCAGCTGT

TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG WO 98/46750 WO 9846750PCTIUS98/0751 I GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG 840

CCC

843 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 813 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCTAACTGCT

TTGCTGGACC

120

CGACTTCCAA

180

ATTGAGGCAA

240

CGACATCCAA

300

TATCTGGTTA

360

CCGGGTGGTG

420

AGCCCCATCT

480

GATTACCCAG

540

CGGCTGGTCC

600

CAAGGCTTGC

660

CCCCCCCCCA

720

TCCGAGCAGC

780

GAGCTGCAGT

SEQUENCE DESCRIPTION: SEQ ID CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC CGAACAACCT CAATGACGAA GACGTCTCTA

ACCTGGAGAG

TTCTTCGTAA

TCATCATCAA.

CCCTTGAGCA

GTTCTGGCGG

CCTCCGACTT

TCACCGTGGC

TGGCACAGCG

TGGAGCGCGT

GCTGTCTTCG

TGGTGGCGCT

GTCAGCCCGA

CTTCGTAAGG

TCTCCAACCA

GGCAGGTGAC

AGCGCAGGAA

CGGCTCCAAC

CGCTGTCAAA

CTCCAACCTG

CTGGATGGAG

GAACACGGAG

CTTCGTCCAG

GAAGCCCTGG

CTCCTCAACC

GCTGTCAAGA

TGTCTGCCCT

TGGCAAGAAT

CAACAGTACG

ATGGCCACCC

ATCCGTGAGC

CAGGACGAGG

CGGCTCAAGA

ATACACTTTG

ACCAACATCT

ATCACTCGCC

CTG

TCCTGATGGA

ACTTAGAAAA

CTGCCACGGC

TCCGGGAAAA

TAGAGGGCGG

AGGACTGCTC

TGTCTGACTA

AGCTCTGCGG

CTGTCGCTGG

TCACCAAATG

CCCGCCTCCT

AGAACTTCTC

ACCTGCACCT

CCGAAACCTT

TGCATCAGGT

CGCACCCTCT

ACTGACGTTC

TGGAGGCTCC

CTTCCAACAC

CCTGCTTCAA

GGCGCTCTGG

GTCCAAGATG

TGCCTTTCAG

GCAGGAGACC

CCGGTGCCTG

INFORMATION FOR SEQ ID NO:16: Wi SEQUENCE CHARACTERISTICS: LENGTH: 858 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT WO 98/46750 TTGCTGGACC CGAACAACCT 120 CGACTTCCAA ACCTGGAGAG 180 ATTGAGGCAA' TTCTTCGTAA

CGACATCCAA

300

TATCTGGTTA

360

CCGGGTGAAC

420

CATAAATCTC

480

GACTTCGCTG,

540

GTGGCCTCCA

600

CAGCGCTGGA

660

CGCGTGAACA

720

CTTCGCTTCG

780

GCGCTGAAGC

840

CCCGACTCCT

858

TCATCATCAA

CCCTTGAGCA

CGTCTGGTCC

CAAACATGGC

TCAAAATCCG

ACCTGCAGGA

TGGAGCGGCT

CGGAGATACA

TCCAGACCAA

CCTGGATCAC

CAACCCTG

CAATGACGAA

CTTCGTAAGG

TCTCCAACCA

GGCAGGTGAC

AGCGCAGGAA

AATCTCTACT

CACCCAGGAC

TGAGCTGTCT

CGAGGAGCTC

CAAGACTGTC

CTTTGTCACC

CATCTCCCGC

TCGCCAGAAC

GACGTCTCTA

GCTGTCAAGA

TGTCTGCCCT

TGGCAAGAAT

CAACAGTACG

ATCAACCCGT

TGCTCCTTCC

GACTACCTGC

TGCGGGGCGC

GCTGGGTCCA

AAATGTGCCT

CTCCTGCAGG

TTCTCCCGGT

TCCTGATGGA

ACTTAGAAAA

CTGCCACGGC

TCCGGGAAAA

TAGAGGGCGG

CTCCTCCGTC

AACACAGCCC

TTCAAGATTA

TCTGGCGGCT

AGATGCAAGG

TTCAGCCCCC

AGACCTCCGA

GCCTGGAGCT

PCT/US98/0751 I

CCGAAACCTT

TGCATCAGGT

CGCACCCTCT

ACTGACGTTC

TGGAGGCTCC

TAAAGAATCT

CATCTCCTCC

CCCAGTCACC

GGTCCTGGCA

CTTGCTGGAG

CCCCAGCTGT

GCAGCTGGTG

GCAGTGTCAG

INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 1047 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCTACACCAT

CAAGTGAGAA

120

AAGCTGTGCC

180

CCCCTGAGCT

240

AGCGGCCTTT

300

GGTCCCACCT

360

CAGATGGAAG

420

TTCGCCTCTG

480

TTCCTGGAGG

540 SEQUENCE DESCRIPTION: TGGGCCCTGC CAGCTCCCTG AGATCCAGGG CGATGGCGCA ACCCCGAGGA GCTGGTGCTG CCTGCCCCAG CCAGGCCCTG TCCTCTACCA GGGGCTCCTG TGGACACACT GCAGCTGGAC AACTGGGAAT GGCCCCTGCC CTTTCCAGCG CCGGGCAGGA TGTCGTACCG CGTTCTACGC SEQ ID NO:17: CCCCAGAGCT TCCTGCTCAA GCGCTCCAGG AGAAGCTGTG CTCGGACACT CTCTGGGCAT CAGCTGGCAG GCTGCTTGAG CAGGCCCTGG AAGGGATATC GTCGCCGACT TTGCCACCAC CTGCAGCCCA CCCAGGGTGC GGGGTCCTGG TTGCTAGCCA CACCTTGCGC AGCCCTACGT

GTCTTTAGAG

TGCCACCTAC

CCCCTGGGCT

CCAACTCCAT

CCCCGAGTTG

CATCTGGCAG

CATGCCGGCC

TCTGCAGAGC

AGAGGGCGGT

WO 98/46750

GGAGGCTCCC

600

AAAGAATCTC

660

ATCTCCTCCG

720

CCAGTCACCG

780

GTCCTGGCAC

840

TTGCTGGAGC

900

CCCAGCTGTC

960

CAGCTGGTGG

1020.

CAGTGTCAGC

1.047

CGGGTGAACC

ATAAATCTCC

TGGCCTCCAA

AGCGCTGGAT

GCGTGAACAC

TTCGCTTCGT

CGCTGAAGCC

CCGACTCCTC

GTCTGGTCCA

AAACATGGCC

CAA.AATCCGT

CCTGCAGGAC

GGAGCGGCTC

GGAGATACAC

CCAGACCAAC

CTGGATCACT

AACCCTG

ATCTCTACTA

ACCCAGGACT

GAGCTGTCTG

GAGGAGCTCT

AAGACTGTCG

TTTGTCACCA

ATCTCCCGCC

CGCCAGAACT

TCAACCCGTC

GCTCCTTCCA

ACTACCTGCT

GCGGGGGCCT

CTGGGTCCAA

AATGTGCCTT

TCCTGCAGGA

TCTCCCGGTG

PCT/US98/0751 1

TCCTCCGTCT

ACACAGCCCC

TCAAGATTAC

CTGGCGGCTG

GATGCAAGGC

TCAGCCCCCC

GACCTCCGAG

CCTGGAGCTG

INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 942 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCCACTCAGG

CGTGAGCTGT

120

GACGAGGAGC

180

CTCAAGACTG

240

CACTTTGTCA

300

AACATCTCCC

360

ACTCGCCAGA

420

TACGTAGAGG

480

CCGTCTCCTC

540

TTCCAACACA

600

CTGCTTCAAG

660

GGCCTCTGGC

720

TCCAAGATGC

780

GCCTTTCAGC

840 SEQUENCE DESCRIPTION: SEQ ID NO:18: ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC

CTGACTACCT

TCTGCGGGGG

TCGCTGGGTC

CCAAATGTGC

GCCTCCTGCA

ACTTCTCCCG

GCGGTGGAGG

CGTCTAAAGA

GCCCCATCTC

ATTACCCAGT

GGCTGGTCCT

AAGGCTTGCT

CCCCCCCCAG

GCTTCAAGAT

CCTCTGGCGG

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

CTCCCCGGGT

ATCTCATAAA

CTCCGACTTC

CACCGTGGCC

GGCACAGCGC

GGAGCGCGTG

CTGTCTTCGC

TACCCAGTCA

CTGGTCCTGG

GGCTTGCTGG

CCCCCCAGCT

GAGCAGCTGG

CTGCAGTGTC

GAACCGTCTG

TCTCCAAACA

GCTGTCAAAA

TCCAACCTGC

TGGATGGAGC

AACACGGAGA

TTCGTCCAGA

CCGTGGCCTC

CACAGCGCTG

AGCGCGTGAA

GTCTTCGCTT

TGGCGCTGAA.

AGCCCGACTC

GTCCAATCTC

TGGCTACCCA

TCCGTGAGCT

AGGACGAGGA

GGCTCAAGAC

TACACTTTGT

CCAACATCTC

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCCTGGATC

CTCAACCCTG

TACTATCAAC

GGACTGCTCC

GTCTGACTAC

GCTCTGCGGG

TGTCGCTGGG

CACCAAATGT

CCGCCTCCTG

WO 98/46750 WO 9846750PCT/US98/07511I CAGGAGACCT CCGAGCAGCT GGTGGCGCTG AAGCCCTGGA TCACTCGCCA GAACTTCTCC 900 CGGTGCCTGG AGCTGCAGTG TCAGCCCGAC TCCTCAACCC TG 942 INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 1047 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCCACCCAGG

CGTGAGCTGT

120

GACGAGGAGC

180

CTCAAGACTG

240

CACTTTGTCA

300

AACATCTCCC

360

ACTCGCCAGA

420

TACGTAGAGG

480

CCGTCTCCTC

540

GCCAGCTCCC

600

GGCGATGGCG

660

GAGCTGGTGC

720

AGCCAGGCCC

780

CAGGGGCTCC

840

CTGCAGCTGG

900

ATGGCCCCTG

960

CGCCGGGCAG

1020

CGCGTTTTAC

1047 SEQUENCE DESCRIPTION: SEQ ID NO:19: ACTGCTCCTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CTGACTACCT

TCTGCGGGGG

TCGCTGGGTC

CCAAATGTGC

GCCTCCTGCA.

ACTTCTCCCG

GCGGTGGAGG

CGTCTAAAGA

TGCCCCAGAG

CAGCGCTCCA

TGCTCGGACA

TGCAGCTGGC

TGCAGGCCCT.

ACGTCGCCGA

CCCTGCAGCC

GAGGGGTCCT

GCCACCTTGC

CCTCTGGCGG

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

CTCCCCGGGT

ATCTCATAAA

CTTCCTGCTC

GGAGAAGCTG

CTCTCTGGGC

AGGCTGCTTG

GGAAGGGATA

CTTTGCCACC

CACCCAGGGT

GGTTGCTAGC

GCAGCCC

CTGGTCCTGG

GGCTTGCTGG

CCC CC CAGCT

GAGCAGCTGG

CTGCAGTGTC

GAACCGTCTG

TCTCCAAACA

AAGTGCTTAG

TGTGCCACCT

ATCCCCTGGG

AGCCAACTCC

TCCCCCGAGT

ACCATCTGGC

GCCATGCCGG

CATCTGCAGA

CACAGCGCTG

AGCGCGTGAA

GTCTTCGCTT

TGGCGCTGAA

AGCCCGACTC

GTCCAATCTC

TGGCTACACC

AGCAAGTGAG

ACAAGCTGTG

CTCCCCTGAG

ATAGCGGCCT

TGGGTCCCAC

AGCAGATGGA

CCTTCGCCTC

GCTTCCTGGA

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCATGGATC

CTCAACCCTG

TACTATCAAC

ATTAGGCCCT

GAAGATCCAG

CCACCCCGAG

CTCCTGCCCC

TTTCCTCTAC

CTTGGACACA

AGAACTGGGA

TGCTTTCCAG

GGTGTCGTAC

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1003 base pairs TYPE: nucleic acid WO 98/46750 WO 9846750PCT/US98/0751 I STRANDEDNESS: single TOPOLOGY: linear (xi) 'SEQUENCE DESCRIPTION: SEQ ID

GGCCACTCAG

CCGTGAGCTG

120

GGACGAGGAG

180

GCTCAAGACT

240

ACACTTTGTC

300

CAACATCTCC

360

CACTCGCCAG

420

GTACGTAGAG

480

TACACCATTG

540

AGTGAGAAAG

600

GCTGTGCCAC

660

CCTGAGCTCC

720

CGGCCTTTTC

780

TCCCACCTTG

840

GATGGAAGAA

900

CGCCTCTGCT

960

CCTGGAGGTG

1003 GACTGCTCTT TTCAACACAG CCCCATCTCC TCCGACTTCG

TCTGACTACC

CTCTGCGGGG

GTCGCTGGGT

ACCAAATGTG

CGCCTCCTGC

AACTTCTCCC

GGCGGTGGAG

GGCCCTGCCA

ATCCAGGGCG

CCCGAGGAGC

TGCCCCAGCC

CTCTACCAGG

GACACACTGC

CTGGGAATGG

TTCCAGCGCC

TCGTACCGCG

TGCTTCAAGA

GCCTCTGGCG

CCAAGATGCA

CCTTTCAGCC

AGGAGACCTC

GGTGCCTGGA

GCTCCCCGGG

GCTCCCTGCC

ATGGCGCAGC

TGGTGC!TGCT

AGGCCCTGCA

GGCTCCTGCA

AGCTGGACGT

CCCCTGCCCT

GGGCAGGAGG

TTCTACGCCA

TTACCCAGTC

GCTGGTCCTG

AGGCTTGCTG

CCCCCCCAGC

CGAGCAGCTG

GCTGCAGTGT

TGGTGGTTCT

CCAGAGCTTC

GCTCCAGGAG

CGGACACTCT

GCTGGCAGGC

GGCCCTGGAA

CGCCGACTTT

GCAGCCCACC

GGTCCTGGTT

CCTTGCGCAG

ACCGTGGCCT

GCACAGCGCT

GAGCGCGTGA

TGTCTTCGCT

GTGGCGCTGA.

CAGCCCGACT

GGCGGCGGCT

CTGCTCAAGT

AAGCTGTGTG

CTGGGCATCC

TGCTTGAGCC

GGGATATCCC

GCCACCACCA

CAGGGTGCCA

GCTAGCCATC

CCG

CTGTCAAAAT

CCAACCTGCA

GGATGGAGCG

ACACGGAGAT

TCGTCCAGAC

AGCCCTGGAT

CCTCAACCCT

CCAACATGGC

CTTTAGAGCA

CCACCTACAA

CCTGGGCTCC

AACTCCATAG

CCGAGTTGGG

TCTGGCAGCA.

TGCCGGCCTT

TGCAGAGCTT

INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 843 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTAACCCT TTGCTGGACC CGAACAACCT CAATTCTGAA GACATGGATA TCCTGATGGA ACGAAACCTT 120 CGAACTCCAA ACCTGCTCGC ATTCGTAAGG GCTGTCAAGC ACTTAGAAAA TGCATCAGGT 180 WO 98/46750 ATTGAGGCAA TTCTTCGTAA 240 CGACATCCAA TCATCATCAA 300 TATCTGGTTA 'CCCTTGAGCA 360 CCGGGTGAAC CGTCTGGTCC 420 CATAAATCTC CAAACATGGC 480 GACTTCGCTG TCAAAATCCG 540 GTGGCCTCCA ACCTGCAGGA 600 CAGCGCTGGA TGGAGCGGCT 660 CGCGTGAACA CGGAGATACA 720 CTTCGCTTCG TCCAGACCAA 780 GCGCTGAAGC CCTGGATCAC 840

CCC

843

TCTCCAACCA

GGCAGGTGAC

AGCGCAGGAA

AATCTCTACT

GACTCAGGAC

TGAGCTGTCT

CGAGGAGCTC

CAAGACTGTC

CTTTGTCACC

CATCTCCCGC

TCGCCAGAAC

TGTCTGCCCT

TGGCAAGAAT

CAACAGTACG

ATCAACCCGT

TGTTCTTTCC

GACTACCTGC

TGCGGGGGCC

GCTGGGTCCA

AAATGTGCCT

CTCCTGCAGG

TTCTCCCGGT

CTGCCACGGC

TCCGGGAAAA

TAGAGGGCGG

CTCCTCCGTC

AACACAGCCC

TTCAAGATTA

TCTGGCGGCT

AGATGCAAGG

TTCAGCCCCC

AGACCTCCGA

GCCTGGAGCT

PCTUS98I0751 I

CGCACCCTCT

ACTGACGTTC

TGGAGGCTCC

TAAAGAATCT

CATCTCCTCC

CCCAGTCACC

GGTCCTGGCA

CTTGCTGGAG

CCCCAGCTGT

GCAGCTGGTG

GCAGTGTCAG

INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 858 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTAACCCT

TTGCTGGACC

120

CGAACTCCAA

180

ATTGAGGCAA

240

CGACATCCAA

300

TATCTGGTTA

360

CCGGGTGAAC

420

CATAAATCTC

480

GACTTCGCTG

540

GTGGCCTCCA

600

CAGCGCTGGA

660

CGAACAACCT

ACCTGCTCGC

TTCTTCGTAA

TCATCATCAA

CCCTTGAGCA

CGTCTGGTCC

CAAACATGGC

TCAAAATCCG

ACCTGCAGGA

TGGAGCGGCT

CAATTCTGAA

ATTCGTAAGG

TCTCCAACCA

GGCAGGTGAC

AGCGCAGGAA

AATCTCTACT

AACCCAGGAC

TGAGCTGTCT

CGAGGAGCTC

CAAGACTGTC

GACATGGATA

GCTGTCAAGC

TGTCTGCCCT

TGGCAAGAAT

CAACAGTACG

ATCAACCCGT

TGCTCTTTTC

GACTACCTGC

TGCGGGGGCC

GCTGGGTCCA

TCCTGATGGA

ACTTAGAAAA

CTGCCACGGC

TCCGGGAAAA

TAGAGGGCGG

CTCCTCCGTC

AACACAGCCC

TTCAAGATTA

TCTGGCGGCT

AGATGCAAGG

ACGAAACCTT

TGCATCAGGT

CGCACCCTCT

ACTGACGTTC

TGGAGGCTCC

TAAAGAATCT

CATCTCCTCC

CCCAGTCACC

GGTCCTGGCA

CTTGCTGGAG

WO 98/46750 CGCGTGAACA CGGAGATACA CTTTGTCACC 720 CTTCGCTTCG TCCAGACCAA CATCTCCCGC 780 GCGCTGAAGC 'CCTGGATCAC TCGCCAGAAC 840 CCCGACTCCT CAACCCTG 858 ID PCT/US98/07511 AAATGTGCCT TTCAGCCCCC CCCCAGCTGT CTCCTGCAGG AGACCTCCGA GCAGCTGGTG TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 843 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCCACTCAGG

CGTGAGCTGT

120

GACGAGGAGC

180

CTCAAGACTG

240

CACTTTGTCA

300

AACATCTCCC

360

ACTCGCCAGA

420

GGAGGCTCCC

480

AAAGAATCTC

540

CATCACTTAA

600

ATGGATATCC

660

GTCAAGCACT

720

CTGCCCTCTG

780

CAAGAATTCC

840

CAG

843 SEQUENCE DESCRIPTION: SEQ ID NO:23: ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC

CTGACTACCT

TCTGCGGGGG

TCGCTGGGTC

CCAAATGTGC

GCCTCCTGC.A

ACTTCTCCCG

CGGGTGAACC

ATAAATCTCC

AGAGACCACC

TGATGGAACG

TAGAA.AATGC

CCACGGCCGC

GGGAAAAACT

GCTTCAAGAT

CCTCTGGCGG

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

GTCTGGTCCA.

AAACATGGCT

TAACCCTTTG

AAACCTTCGA

ATCAGGTATT

ACCCTCTCGA

GACGTTCTAT

TACCCAGTCA

CTGGTCCTGG

GGCTTGCTGG

CCCCCCAGCT

GAGCAGCTGG

CTGCAGTGTC

ATCTCTACTA

AACTGCTCTA

CTGGACCCGA

ACTCCAAACC

GAGGC!AATTC

CATCCAATCA

CTGGTTACCC

CCGTGGCCTC

CACAGCGCTG

AGCGCGTGAA

GTCTTCGCTT

TGGCGCTGAA

AGCCCTACGT

TCAACCCGTC

TAATGATCGA

ACAACCTCAA

TGCTCGCATT

TTCGTAATCT

TCATCAAGGC

TTGAGCAAGC

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCCTGGATC

AGAGGGCGGT

TCCTCCGTCT

TGAAATTATA

TTCTGAAGAC

CGTAAGGGCT

CCAACCATGT

AGGTGACTGG

GCAGGAACAA

INFORMATION FOR SEQ ID, NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 858 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear WO 98/46.750 WO 9846750PCTUS98/0751 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:

GCCACCCAGG

CGTGAGCTGT

120

GACGAGGAGC

180

CTCAAGACTG

240

CACTTTGTCA

300

AACATCTCCC

360

ACTCGCCAGA

420

TACGTAGAGG

480

CCGTCTCCTC

540

ATCGATGAAA

600

CTCAATTCTG

660

GCATTCGTAA

720

AATCTCCAAC

780

AAGGCAGGTG

840

CAAGCGCAGG

858 ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC

CTGACTACCT

TCTGCGGGGG

TCGCTGGGTC

CCAAATGTGC

GCCTCCTGCA

ACTTCTCCCG

GCGGTGGAGG

CGTCTAAAGA

TTATACATCA

AAGACATGGA

GGGCTGTCAA

CATGTCTGCC

ACTGGCAAGA

AACAACAG

GCTTCAAGAT

CCTCTGGCGG

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

CTCCCCGGGT

ATCTCATAAA

CTTAAAGAGA

TATCCTGATG

GCACTTAGAA

CTCTGCCACG

ATTCCGGGAA

TACCCAGTCA

CTGGTCCTGG

GGCTTGCTGG

CCCCCCAGCT

GAGCAGCTGG

CTGCAGTGTC

GAACCGTCTG

TCTCCAAACA

CCACCTAACC

GAACGAAACC

AATGCATCAG

GCCGCACCCT

AAACTGACGT

CCGTGGCCTC

CACAGCGCTG

AGCGCGTGAA

GTCTTCGCTT

TGGCGCTGA.A

AGCCCGACTC

GTCCAATCTC

TGGCTAACTG

CTTTGCTGGA

TTCGAACTCC

GTATTGAGGC

CTCGACATCC

TCTATCTGGT

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCCTGGATC

CTCAACCCTG

TACTATCAAC

CTCTATAATG

CCCGAACAAC

AAACCTGCTC

AATTCTTCGT

AATCATCATC

TACCCTTGAG

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 939 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCCACTCAGG

CGTGAGCTGT

120

GACGAGGAGC

180

CTCAAGACTG

240

CACTTTGTCA

300

AACATCTCCC

360 SEQUENCE DESCRIPTION: ACTGCTCTTT TCAACACAGC CTGACTACCT GCTTCAAGAT TCTGCGGGGG CCTCTGGCGG TCGCTGGGTC CAAGATGCAA CCAA.ATGTGC CTTTCAGCCC GCCTCCTGCA GGAGACCTCC SEQ ID CCCATCTCCT CCGACTTCGC TACCCAGTCA CCGTGGCCTC CTGGTCCTGG CACAGCGCTG GGCTTGCTGG AGCGCGTGAA CCCCCCAGCT GTCTTCGCTT GAGCAGCTGG TGGCGCTGAA

TGTCAAAATC

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCCTGGATC

WO 98/46750 ACTCGCCAGA ACTTCTCCCG 420 TACGTAGAGG GCGGTGGAGG 480 TCTCCGGCGC 'CGCCTGCTTG 540 GTCCTTCACA GCAGACTGAG 600 CTGCCTGCTG TGGACTTTAG 660 CAGGACATTC TGGGAGCAGT 720 CTGGGACCCA CTTGCCTCTC 780 CTTGGGGCCC TGCAGAGCCT 840 CACAAGGATC CCAATGCCAT 900 TTCCTGATGC TTGTAGGAGG 939

GTGCCTGGAG

CTCCCCGGGT

TGACCTCCGA

CCAGTGCCCA

CTTGGGAGAA

GACCCTTCTG

ATCCCTCCTG

CCTTGGAACC

CTTCCTGAGC

GTCCACCCTC

CTGCAGTGTC

GGTGGTTCTG

GTCCTCAGTA.

GAGGTTCACC

TGGAAAACCC

CTGGAGGGAG

GGGCAGCTTT

CAGCTTCCTC

TTCCAACACC

TGCGTCAGG

AGCCCGACTC

GCGGCGGCTC

AACTGCTTCG

CTTTGCCTAC

AGATGGAGGA

TGATGGCAGC

CTGGACAGGT

CACAGGGCAG

TGCTCCGAGG

PCTIUS98/07511I

CTCAACCCTG

CAACATGGCG

TGACTCCCAT

ACCTGTCCTG

GACCAAGGCA

ACGGGGACAA

CCGTCTCCTC

GACCACAGCT

AAAGGTGCGT

INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 996 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCGTCCCCAG

CATGTCCTTC

120

CTGCTGCCTG

180

GCACAGGACA

240

CAACTGGGAC

300

CTCCTTGGGG

360

GCTCACAAGG

420

CGTTTCCTGA

480

GAGGGCGGTG

540

CCTCCGTCTA

600

CACAGCCCCA

660

CAAGATTACC

720

TGGCGGCTGG

780 SEQUENCE DESCRIPTION: SEQ ID NO:26: CTCCACCTGC TTGTGACCTC CGAGTCCTCA GTAAACTGCT TCGTGACTCC

ACAGCAGACT

CTGTGGACTT

TTCTGGGAGC

CCACTTGCCT

CCCTGCAGAG

ATCCCAATGC

TGCTTGTAGG

GAGGCTCCCC

AAGAATCTCA

TCTCCTCCGA

CAGTCACCGT

TCCTGGCACA

GAGCCAGTGC

TAGCTTGGGA

AGTGACCCTT

CTCATCCCTC

CCTCCTTGGA

CATCTTCCTG

AGGGTCCACC

GGGTGAACCG

TAAATCTCCA

CTTCGCTGTC

GGCCTCCAAC

GCGCTGGATG

CCAGAGGTTC

GAATGGAAAA

CTGCTGGAGG

CTGGGGCAGC

ACCCAGCTTC

AGCTTCCAAC

CTCTGCGTCA

TCTGGTCCAA

AACATGGCTA

AAAATCCGTG

CTGCAGGACG

GAGCGGCTCA

ACCCTTTGCC

CCCAGATGGA

GAGTGATGGC

TTTCTGGACA

CTCCACAGGG

ACCTGCTCCG

GGGAATTCCA

TCTCTACTAT

CCCAGGACTG

AGCTGTCTGA

AGGAGCTCTG

AGACTGTCGC

TACACCTGTC

GGAGACCAAG

AGCACGGGGA

GGTCCGTCTC

CAGGACCACA

AGGAAAGGTG

TGCATACGTA

CAACCCGTCT

CTCCTTCCAA

CTACCTGCTT

CGGGGGCCTC

TGGGTCCAAG

WO 98/46750 WO 9846750PCTJUS98IO751 1 ATGCAAGGCT TGCTGGAGCG CGTGAACACG 840 CAGCCCCCCC CCAGCTGTCT TCGCTTCGTC 900 ACCTCCGAGC 'AGCTGGTGGC GCTGAAGCCC 960 CTGGAGCTGC AGTGTCAGCC CGACTCCTCA 996 GAGATACACT TTGTCACCAA ATGTGCCTTT CAGACCAACA TCTCCCGCCT CCTGCAGGAG TGGATCACTC GCCAGAACTT CTCCCGGTGC

ACCCTG

INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 1020 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC CGTGAGCTGT CTGACTACCT 120

GACGAGGAGC

180

CTCAAGACTG

240

CACTTTGTCA

300

AACATCTCCC

360

ACTCGCCAGA

420

TACGTAGAGG

480

CCGTCTCCTC

540

TGTGACAGCC

600

ACGGGCTGTG

660

AATTTCTATG

720

CTGGCCCTGC

780

CCGTGGGAGC

840

ACTCTGCTTC

900

GCTGCTCCAC

960

AATTTCCTCC

1020

TCTGCGGGGG

TCGCTGGGTC

CCAAATGTGC

GCCTCCTGCA

ACTTCTCCCG

GCGGTGGAGG

CGTCTAAAGA

GAGTCCTGGA

CTGAACACTG

CCTGGAAGAG

TGTCGG.AAGC

CCCTGCAGCT

GGGCTCTGCG

TCCGA.ACAAT

GGGGAAAGCT

GCTTCAAGAT

CCTCTGGCGG

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

CTCCCCGGGT

ATCTCATAAA

GAGGTACCTC

CAGCTTGAAT

GATGGAGGTC

TGTCCTGCGG

GCATGTGGAT

AGCCCAGAAG

CACTGCTGAC

GAAGCTGTAC

TACCCAGTCA

CTGGTCCTGG

GGCTTGCTGG

CCCCCCAGCT

GAGCAGCTGG

CTGCAGTGTC

GAACCGTCTG

TCTCCAAACA

TTGGAGGCCA

GAGAATATCA

GGGCAGCAGG

GGCCAGGCCC

AAAGCCGTCA

GAAGCCATCT

ACTTTCCGCA

ACAGGGGAGG

CCGTGGCCTC

CACAGCGCTG

AGCGCGTGAA

GTCTTCGCTT

TGGCGCTGAA

AGCCCGACTC

GTCCAATCTC

TGGCCCCACC

AGGAGGCCGA

CTGTCCCAGA

CCGTAGAAGT

TGTTGGTCAA.

GTGGCCTTCG

CCCCTCCAGA

A.ACTCTTCCG

CCTGCAGGAC

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCCTGGATC

CTCAACCCTG

TACTATCAAC

ACGCCTCATC

GAATATCACG

CACCAAAGTT

CTGGCAGGGC

CTCTTCCCAG

CAGCCTCACC

TGCGGCCTCA

AGTCTACTCC

AGGGGACAGA

INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: WO 98/46750 WO 9846750PCTIUS98/0751 1 LENGTH: 975 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCCCCACCAC

GAGGCCGAGA

120

GTCCCAGACA

180

GTAGAAGTCT

240

TTGGTCAACT

300

GGCCTTCGCA

360

CCTCCAGATG

420

CTCTTCCGAG

480

TGCAGGACAG

540

GGCGGCTCCA

600

TTCGCTGTCA

660

GCCTCCAACC

720

CGCTGGATGG

780

GTGAACACGG

840

CGCTTCGTCC

900

CTGAAGCCCT

960

GACTCCTCAA

975 SEQUENCE DESCRIPTION: SEQ ID NO:28: GCCTCATCTG TGACAGCCGA GTCCTGGAGA

ATATCACGAC

CCAAAGTTAA

GGCAGGGCCT

CTTCCCAGCC

GCCTCACCAC

CGGCCTCAGC

TCTACTCCAA

GGGACAGATA

ACATGGCCAC

AAATCCGTGA

TGCAGGACGA

AGCGGCTCAA

AGATACACTT

AGACCAACAT

GGATCACTCG

CCCTG

GGGCTGTGCT

TTTCTATGCC

GGCCCTGCTG

GTGGGAGCCC

TCTGCTTCGG

TGCTCCACTC

TTTCCTCCGG

CGTAGAGGGC

TCAGGACTGC

GCTGTCTGAC

GGAGCTCTGC

GACTGTCGCT

TGTCACCAAA

CTCCCGCCTC

GAACACTGCA

TGGAAGAGGA

TCGGAAGCTG

CTGCAGCTGC

GCTCTGCGAG

CGAACAATCA

GGAAAGCTGA

GGTGGAGGCT

TCTTTTCAAC

TACCTGCTTC

GGGGGCCTCT

GGGTCCAAGA

TGTGCCTTTC

CTGCAGGAGA

GGTACCTCTT

GCTTGAATGA

TGGAGGTCGG

TCCTGCGGGG

ATGTGGATA.A

CCCAGAAGGA

CTGCTGACAC

AGCTGTACAC

CCCCGGGTGG

ACAGCCCCAT

AAGATTACCC

GGCGGCTGGT

TGCAAGGCTT

AGCCCCCCCC

CCTCCGAGCA

TGGAGCTGCA

GGAGGCCAAG

GAATATCACT

GCAGCAGGCC

CCAGGCCCTG

AGCCGTCAGT

AGCCATCTCC

TTTCCGCAAA

AGGGGAGGCC

TGGTTCTGGC

CTCCTCCGAC

AGTCACCGTG

CCTGGCACAG

GCTGGAGCGC

CAGCTGTCTT

GCTGGTGGCG

GTGTCAGCCC

CCAGAACTTC TCCCGGTGCC INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 28 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: GTAGTCCATG GCCACCCAGG ACTGCTCC INFORMATION FOR SEQ ID WO 98/46750 PCT/US98/07511 SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID GCATTACGTA GGGCTGACAC TGCAGCTCCA G 31 INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: GCATTACGTA CAGGGTTGAG GAGTCGGGCT G 31 INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 44 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: CCTGCAGGAC AGGGGACAGA TACGTAGAGG GCGGTGGAGG CTCC 44 INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 48 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: CCGGGGAGCC TCCACCGCCC TCTACGTATC TGTCCCCTGT CCTGCAGG 48 INFORMATION FOR SEQ ID NO:34: WO 98/46750PC/S805 PCTIUS98/07511 SEQUENCE CHARACTERISTICS: LENGTH: 266 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: Ala Asn Cys Ser Ile Met Ile Asp Giu Ile 1 Pro Ser Val Leu Arg Lys Tyr Ser Ser 145 Asp Gly Lys Thr Cys 225 Ser Ser Pro Ile Arg Arg His Leu Val Asn 130 Asp Tyr Ala Thr Giu 210 Leu Giu Arg Ala Leu Ala Asn Pro Thr Giu 115 Met Phe Pro Leu Val 195 Ile Arg Gin Cys Pro Met Val Leu Ile Phe 100 Gly Ala Ala Val Trp 180 Ala His Phe Leu Leu 260 5 Leu Asp Lys Gin Ile Tyr Gly Thr Val Thr 165 Arg Gly Phe Val Val 245 Giu Leu Arg Asn Pro 70 Ile Leu Gly Gin Lys 150 Val Leu Ser Val Gin 230 Ala Leu Asp Asn Leu 55 Cys Lys Val Gly Asp 135 Ile Ala Val Lys Thr 215 Thr Leu Gin Pro Leu 40 Giu Leu Ala Thr Ser 120 Cys Arg Ser Leu Met 200 Lys Asn Lys Cys Asn 25 Arg Asn Pro Gly Leu 105 Pro Ser Giu Asn Ala 185 Gin Cys Ile Pro Gin 265 10 Asn Leu Al a Ser Asp 90 Giu Gly Phe Leu Leu 170 Gin Gly Ala Ser Trp 250 Pro Ile Leu Pro Ser Ala 75 Trp Gin Gly Gin Ser 155 Gin Arg Leu Phe Arg 235 Ile His Asn Asn Gly Thr Gin Ala G ly His 140 Asp Asp Trp Leu Gin 220 Leu Thr His Asp Leu Ile Ala Giu Gin Ser 125 Ser Tyr Glu Met Giu 205 Pro Leu Arg Leu Glu Giu Glu Ala Phe Giu 110 Gly Pro Leu Giu Giu 190 Arg Pro Gin Gin Lys Asp Ser Ala Pro Arg Gin Gly Ile Leu Leu 175 Arg Val Pro Glu Asn 255 Arg Val Phe Ile Ser Giu Gin Gly Ser Gin 160 Cys Leu Asn Ser Thr 240 Phe INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 281 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg WO 98/46750 Pro Ser Val Leu Arg Lys Tyr Ser Asn 145 Asp Tyr Ala Thr Glu 225 Leu Glu Arg Ala Leu Ala Asn Pro Thr Glu 115 Ile Ala Ala Val Trp 195 Ala His Phe Leu Leu Pro Met Val Leu Ile Phe 100 Gly Asn Thr Val Thr 180 Arg Gly Phe Val Val 260 Glu Leu Asp Lys Gin Ile Tyr Gly Pro Gin Lys 165 Val Leu Ser Val Gin 245 Ala Leu Leu Arg Asn Pro 70 Ile Leu Gly Ser Asp 150 Ile Ala Val Lys Thr 230 Thr Leu Gin Asp Asn Leu Cys Lys Val Gly Pro 135 Cys Arg Ser Leu Met 215 Lys Asn Lys Cys Pro Leu 40 Glu Leu Ala Thr Ser 120 Pro Ser Glu Asn Ala 200 Gin Cys Ile Pro Gin 83 Asn 25 Arg Asn Pro Gly Leu 105 Pro Ser Phe Leu Leu 185 Gin Gly Ala Ser Trp 265 Pro PCT/US98/07511 10 Asn Leu Ala Ser Asp 90 Glu Gly Lys Gin Ser 170 Gin Arg Leu Phe Arg 250 Ile Leu Pro Ser Ala 75 Trp Gin Glu Glu His 155 Asp Asp Trp Leu Gin 235 Leu Thr Asn Asn Gly Thr Gin Ala Pro Ser 140 Ser Tyr Glu Met Glu 220 Pro Leu Arg Asp Leu Ile Ala Glu Gin Ser 125 His Pro Leu Glu Glu 205 Arg Pro Gin Gin Glu Glu Glu Ala Phe Glu 110 Gly Lys Ile Leu Leu 190 Arg Val Pro Glu Asn 270 Asp Ser Ala Pro Arg Gin Pro Ser Ser Gin 175 Cys Leu Asn Ser Thr 255 Phe Val Phe Ile Ser Glu Gin Ile Pro Ser 160 Asp Gly Lys Thr Cys 240 Ser Ser INFORMATION FOR SEQ ID NO:36: SEQUENCE CHARACTERISTICS: LENGTH: 271 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Asn Cys Ser Ile Met Ile Asp Glu Ile 1 5 10 Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn 25 Ser Ile Leu Met Asp Arg Asn Leu Arg Leu 40 Val Arg Ala Val Lys Asn Leu Glu Asn Ala 55 Leu Arg Asn Leu Gin Pro Cys Leu Pro Ser 70 NO:36: Ile His His Leu Lys Arg Leu Asn Asp Glu Asp Val Pro Asn Leu Glu Ser Phe Ser Gly Ile Glu Ala Ile Ala Thr Ala Ala Pro Ser 75 WO 98/46750 Arg Lys Tyr Ser Ser 145 Asp Gly Lys Thr Cys 225 Ser Ser His Leu Vai Asn 130 Asp Tyr Ala Thr Giu 210 Leu Giu Arg Pro Thr Gld 115 Met Phe Pro Leu Val 195 Ile Arg Gin Cys Ile Phe 100 Gly Ala Ala Vai Trp 180 Ala His Phe Leu Leu Ile Leu Giy Gin Lys 150 Val Leu Ser Vai Gin 230 Ala Leu Ala Thr Ser 120 Cys Arg Ser Leu Met 200 Lys Asn Lys Cys 84 Gly Leu 105 Pro Ser Giu Asn Ala 185 Gin Cys Ile Pro Gin 265 PCTIUS98/0751 I Asp Giu Gly Phe Leu Leu 170 Gin Giy Ala Ser Trp 250 Pro Trp Gin Gly Gin Ser 155 Gin Arg Leu Phe Arg 235 Ile Asp Gin Ala Gly His 140 Asp Asp Trp Leu Gin 220 Leu Thr Ser Giu Gin Ser 125 Ser Tyr Giu Met Giu 205 Pro Leu Arg Ser Phe Giu 110 Gly Pro Leu G iu Giu 190 Arg Pro Gin Gin Thr 270 Giu Gin Giy Ser Gin 160 Cys Leu Asn Ser Thr 240 Phe INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 286 amino acids TYPE: amino acid STRANflEDNESS: singie TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Ala Asn Cys Ser Ile Met Ile Asp Giu Ile 1 Pro Ser Val Leu Arg Lys Tyr Ser Asn 145 Asp Pro Ile Arg Arg His Leu Vai Thr 130 Met Phe Aia Leu Ala Asn Pro Thr Giu 115 Ile Ala Ala Pro Met Val Leu Ile Phe 100 Gly Asn Thr Val 5 Leu Asp Lys Gin Ile Tyr Gly Pro Gin Lys Leu Arg Asn Pro 70 Ile Leu Gly Ser Asp 150 Ile Asp Asn Leu 55 Cys Lys Vai Gly Pro 135 Cys Arg Pro Leu 40 Giu Leu Aia Thr Ser 120 Pro Ser Giu Asn 25 Arg Asn Pro Gly Leu 105 Pro Ser Phe Leu 10 Asn Leu Ala Ser Asp 90 Giu Gly Lys Gin Ser Ile Leu Pro Ser Ala Trp Gin Giu Giu His 155 Asp His Asn Asn Giy Thr Gin Ala Pro Ser 140 Ser Tyr His Asp Leu Ile Aia Giu Gin Ser 125 His Pro Leu Leu Giu Giu Glu Ala Phe Giu 110 Giy Lys Ile Leu Lys Asp Ser Ala Pro Arg Gin Pro Ser Ser Gin WO 98/46750 PCT/US98/07511 165 170 175 Tyr Pro Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly 180 185 190 Ala Leu Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys 195 200 205 Thr Val Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr 210 215 220 Glu Ile His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys 225 230 235 240 Leu Arg Phe Val Gin Thr Asn Ile Ser Arg Leu Leu Gin Glu Thr Ser 245 250 255 Glu Gin Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gin Asn Phe Ser 260 265 270 Arg Cys Leu Glu Leu Gin Cys Gin Pro Asp Ser Ser Thr Leu 275 280 285 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 349 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu 1 5 10 Lys Ser Leu Glu Gin Val Arg Lys Ile Gin Gly Asp Gly Ala Ala Leu 25 Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 40 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 55 Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His 70 75 Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly Ile 90 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala 100 105 110 Asp Phe Ala Thr Thr Ile Trp Gin Gin Met Glu Glu Leu Gly Met Ala 115 120 125 Pro Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala 130 135 140 Phe Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser 145 150 155 160 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Tyr 165 170 175 Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser 180 185 190 Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn 195 200 205 Met Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro Ile Ser Ser Asp 210 215 220 Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr 225 230 235 240 WO 98/46750 86 PCT/US98/07511 Pro Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly 245 250 255 Leu Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr 260 265 270 Val Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu 275 280 285 Ile His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu 290 295 300 Arg Phe Val Gin Thr Asn Ile Ser Arg Leu Leu Gin Glu Thr Ser Glu 305 310 315 320 Gin Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gin Asn Phe Ser Arg 325 330 335 Cys Leu Glu Leu Gin Cys Gin Pro Asp Ser Ser Thr Leu 340 345 INFORMATION FOR SEQ ID NO:39: SEQUENCE CHARACTERISTICS: LENGTH: 314 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39: Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro Ile Ser Ser Asp Phe 1 5 10 Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro 25 Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu 40 Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val 55 Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu Ile 70 75 His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg 90 Phe Val Gin Thr Asn Ile Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin 100 105 110 Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gin Asn Phe Ser Arg Cys 115 120 125 Leu Glu Leu Gin Cys Gin Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly 130 135 140 Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn 145 150 155 160 Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr 165 170 175 Gin Asp Cys Ser Phe Gin His Ser Pro Ile Ser Ser Asp Phe Ala Val 180 185 190 Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro Val Thr 195 200 205 Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg 210 215 220 Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly 225 230 235 240 Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe WO 98/46750PCISI751 PCT/US98/07511 Val Gin Ala Leu 305 Tb Tb Le 29 Gi 245 250 .r Lys Cys Ala Phe Gin Pro Pro Pro Se 260 265 r Asn Ile Ser Arg Leu Leu Gin Glu Th 275 280 u Lys Pro Trp Ile Thr Arg Gin Asn Ph 0 295 n Cys Gin Pro Asp Ser Ser Thr Leu 310 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 349 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear 255 Cys Leu Arg Phe Val 270 r Ser Giu Gin Leu Val 285 e Ser Arg Cys Leu Glu 300 (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Thr Gin Asp Cys Ser Phe Gin His 1 Ala Val Trp, Ala His Phe Leu Leu Gly 145 Pro Pro Leu Lys Leu 225 Ser Leu Glu Val Thr Arg Gly Phe Val Val Glu 130 Gly Ser Leu Glu Leu 210 Gly Gin Phe Leu Lys Val Leu Ser Val Gin Ala 115 Leu Gly Pro Gly Gin 195 Cys His Ala Leu Gly 275 Ile Ala Val1 Lys Thr Thr 100 Leu Gin Ser Pro Pro 180 Val Al a Ser Leu Tyr 260 Pro 5 Arg Ser Leu Met Lys Asn Lys Cys Pro Ser 165 Ala Arg Thr Leu Gin 245 Gin Thr Glu Asn Ala Gin 70 Cys Ile Pro Gin Gly 150 Lys Ser Lys Tyr Gly 230 Leu Gly Leu Leu Leu Gin 55 Gly Ala Ser Trp Pro 135 Giu Glu Ser Ile Lys 215 Ile Ala Leu Asp Ser Gin 40 Arg Leu Phe Axg Ile 120 Asp Pro Ser Leu Gin 200 Leu Pro Gly Leu Thr 280 Asp Asp Trp, Leu Gin Leu 105 Thr Ser Ser His Pro 185 Gly Cys Trp Cys Gin 265 Leu Ser Tyr Glu Met Glu Pro Leu A-rg Ser Gly Lys 170 Gin Asp His Ala Leu 250 Ala Gin Pro Leu Glu Glu Arg 75 Pro Gin Gin Thr Pro 155 Ser Ser Gly Pro Pro 235 Ser Leu Leu Ile Leu Leu Arg Val Pro Giu Asn Leu 140 Ile Pro Phe Ala Giu 220 Leu Gin Giu Asp Ser Gin Cys Leu Asn Ser Thr Phe 125 Tyr Ser Asn Leu Aia 205 Giu Ser Leu Gly Val 285 Phe Pro Leu Val Ile Arg Gin Cys Gly Asn 160 Thr Cys Glu Leu Pro 240 Gly Pro Phe WO 98/46750 PCT/US98/07511 Ala Thr Thr Ile Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro Ala 290 295 300 Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gin 305 310 315 320 Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe Leu 325 330 335 Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 340 345 INFORMATION FOR SEQ ID NO:41: SEQUENCE CHARACTERISTICS: LENGTH: 334 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41: Ala Thr Gin Asp Cys 1 Ala Val Trp Ala His Phe Leu Leu Gly 145 Thr Ser Glu Leu Pro 225 Gly Pro Phe Val Thr Arg Gly Phe Val Val Glu 130 Gly Pro Leu Lys Leu 210 Ser Leu Glu Ala Lys Val Leu Ser Val Gin Ala 115 Leu Gly Leu Glu Leu 195 Gly Gin Phe Leu Thr Ile Ala Val Lys Thr Thr 100 Leu Gin Ser Gly Gin 180 Cys His Ala Leu Gly 260 Thr 5 Arg Ser Leu Met Lys Asn Lys Cys Pro Pro 165 Val Ala Ser Leu Tyr 245 Pro Ile Ser Glu Asn Ala Gin 70 Cys Ile Pro Gin Gly 150 Ala Arg Thr Leu Gin 230 Gin Thr Trp Phe Leu Leu Gin Gly Ala Ser Trp Pro 135 Gly Ser Lys Tyr Gly 215 Leu Gly Leu Gin Gin Ser Gin 40 Arg Leu Phe Arg Ile 120 Asp Gly Ser Ile Lys 200 Ile Ala Leu Asp Gin His Asp 25 Asp Trp Leu Gin Leu 105 Thr Ser Ser Leu Gin 185 Leu Pro Gly Leu Thr 265 Met Ser 10 Tyr Glu Met Glu Pro 90 Leu Arg Ser Gly Pro 170 Gly Cys Trp Cys Gin 250 Leu Glu Pro Leu Glu Glu Arg 75 Pro Gin Gin Thr Gly 155 Gin Asp His Ala Leu 235 Ala Gin Glu Ser Gin Cys Leu Asn Ser Thr Phe 125 Tyr Ser Phe Ala Glu 205 Leu Gin Glu Asp Gly Ser Asp Gly Lys Thr Cys Ser 110 Ser Val Asn Leu Ala 190 Glu Ser Leu Gly Val 270 Met Asp Tyr Gly Thr Glu Leu Glu Arg Glu Met Leu 175 Leu Leu Ser His Ile 255 Ala Ala Phe Pro Leu Val Ile Arg Gin Cys Gly Ala 160 Lys Gin Val Cys Ser 240 Ser Asp Pro 275 280 285 Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe WO 98/46750 PCT/US98/07511 290 295 300 Gin Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gin Ser Phe 305 310 315 320 Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro 325 330 INFORMATION FOR SEQ ID NO:42: SEQUENCE CHARACTERISTICS: LENGTH: 281 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: Ala Asn Cys Ser Ile Met Ile Asp Glu Ile 1 Pro Pro Asp Ile Val Arg Leu Arg Arg His Lys Leu Tyr Val Ser Thr 130 Asn Met 145 Asp Phe Tyr Pro Gly Leu Thr Val 210 Glu Ile 225 Leu Arg Glu Gin Arg Cys Asn Leu Ala Asn Pro Thr Glu 115 Ile Ala Ala Val Trp 195 Ala His Phe Leu Leu Pro Met Val Leu Ile Phe 100 Gly Asn Thr Val Thr 180 Arg Gly Phe Val Val 260 Glu 5 Leu Glu Lys Gin Ile Tyr Gly Pro Gin Lys 165 Val Leu Ser Val Gin 245 Ala Leu Leu Arg His Pro 70 Ile Leu Gly Ser Asp 150 Ile Ala Val Lys Thr 230 Thr Leu Gin Asp Asn Leu 55 Cys Lys Val Gly Pro 135 Cys Arg Ser Leu Met 215 Lys Asn Lys Cys Pro Leu 40 Glu Leu Ala Thr Ser 120 Pro Ser Glu Asn Ala 200 Gin Cys Ile Pro Gin 280 Asn 25 Arg Asn Pro Gly Leu 105 Pro Ser Phe Leu Leu 185 Gin Gly Ala Ser Trp 265 Pro 10 Asn Thr Ala Ser Asp 90 Glu Gly Lys Gin Ser 170 Gin Arg Leu Phe Arg 250 Ile Ile Leu Pro Ser Ala 75 Trp Gin Glu Glu His 155 Asp Asp Trp Leu Gin 235 Leu Thr His Asn Asn Gly Thr Gin Ala Pro Ser 140 Ser Tyr Glu Met Glu 220 Pro Leu Arg His Ser Leu Ile Ala Glu Gin Ser 125 His Pro Leu Glu Glu 205 Arg Pro Gin Gin Leu Glu Leu Glu Ala Phe Glu 110 Gly Lys Ile Leu Leu 190 Arg Val Pro Glu Asn 270 Lys Asp Ala Ala Pro Arg Gin Pro Ser Ser Gin 175 Cys Leu Asn Ser Thr 255 Phe Arg Met Phe Ile Ser Glu Gin Ile Pro Ser 160 Asp Gly Lys Thr Cys 240 Ser Ser INFORMATION FOR SEQ ID NO:43: SEQUENCE CHARACTERISTICS: WO 98/46750 WO 9846750PCTIUS98/0751 I LENGTH: 286 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43: Ala Asn Cys Ser Ile Met Ile 1 Pro Asp Val Leu Arg Lys Tyr Ser Asn 145 Asp Tyr Gly Thr Giu 225 Leu Glu Arg Pro Ile Arg Arg His Leu Val Thr 130 Met Phe Pro Leu Val 210 Ile Arg Gin Cys Asn Leu Ala Asn Pro Thr Glu 115 Ile Ala Ala Val Trp, 195 Ala His Phe Leu Leu 275 Pro Met Val Leu Ile Phe 100 Gly Asn Thr Val Thr 180 Arg Gly Phe Val Val 260 Glu 5 Leu Giu Lys Gin Ile Tyr Gly Pro Gin Lys 165 Val Leu Ser Val Gin 245 Ala Leu Leu Arg His Pro 70 Ile Leu Gly Ser Asp 150 Ile Ala Val Lys Thr 230 Thr Leu Gin Asp Asn Leu 55 Cys Lys Val Gly Pro 135 Cys Arg Ser Leu Met 215 Lys Asn Lys Cys Asp Pro Leu 40 Giu Leu Ala Thr Ser 120 Pro Ser Giu Asn Ala 200 Gin Cys Ile Pro Gin 280 Gu Ile Ile 10 Asn Asn Leu 25 Arg Thr Pro Asn Ala Ser Pro Ser Ala 75 Gly Asp Trp, 90 Leu Giu Gln 105 Pro Giy Glu Ser Lys Glu Phe Gin His 155 Leu Ser Asp 170 Leu Gin Asp 185 Gin Arg Trp Gly Leu Leu Ala Phe Gin 235 Ser Arg Leu 250 Trp Ile Thr 265 Pro Asp Ser His Asn Asn Gly Thr Gin Ala Pro Ser 140 Ser Tyr Giu Met Giu 220 Pro Leu Arg Ser His Ser Leu Ile Ala Giu Gin Ser 125 His Pro Leu Giu Giu 205 Arg Pro Gin Gin Thr 285 Leu Giu Leu Giu Ala Phe Giu 110 Gly Lys Ile Leu Leu 190 Arg Val Pro Giu Asn 270 Leu Lys Asp Ala Ala Pro Arg Gin Pro Ser Ser Gin 175 Cys Leu Asn Ser Thr 255 Phe Arg Met Phe Ile Ser Giu Gin Ile Pro Ser 160 Asp Gly Lys Thr Cys 240 Ser Ser INFORMATION FOR SEQ ID NO:44: SEQUENCE CHARACTERISTICS: LENGTH: 281 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: WO 98/46750 WO 9846750PCTIUS98/07511I Ala Ala Val Trp Ala His Phe Leu Leu Gly 145 Lys Asp Pro Leu Glu 225 Leu Ala Thr Thr Val Thr Arg Gly Phe Val Val Giu 130 Glu Glu Glu Asn Arg 210 Asn Pro Gly Leu Gin Lys Val Leu Ser Val Gin Ala 115 Leu Pro Ser Ile Asn 195 Thr Ala Ser Asp Glu 275 Asp Ile Ala Val Lys Thr Thr 100 Leu Gin Ser His Ile 180 Leu Pro Ser Ala Trp 260 Gin Cys Arg Ser Leu Met Lys Asn Lys Cys Gly Lys 165 His Asn Asn Gly Thr 245 Gin Ala Ser Giu Asn Ala Gin 70 Cys Ile Pro Gin Pro 150 Ser His Ser Leu Ile 230 Ala Glu Gin Phe Leu Leu Gin 55 Gly Ala Ser Trp Pro 135 Ile Pro Leu Giu Leu 215 Giu Ala Phe Giu Gin Ser Gin 40 Arg Leu Phe Arg Ile 120 Tyr Ser Asn Lys Asp 200 Ala Ala Pro Arg Gin His Asp Asp Trp Leu Gin Leu 105 Thr Val Thr Met Arg 185 Met Phe Ile Ser Glu 265 Gin Ser 10 Tyr Giu Met Giu Pro 90 Leu Arg Giu Ile Al a 170 Pro Asp Val Leu Arg 250 Lys Pro Leu Glu Glu Arg Pro Gin Gin Gly Asn 155 Asn Pro Ile Arg Arg 235 His Leu Ile Leu Leu Arg Val Pro Glu Asn Gly 140 Pro Cys Asn Leu Ala 220 Asn.

Pro Thr Ser Gin Cys Leu Asn Ser Thr Phe 125 Gly Ser Ser Pro Met 205 Val Leu Ile Phe Asp Tyr Gly Thr Giu Leu Giu Arg S er Pro Met 175 Leu Arg His Pro Ile 255 Leu INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 286 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro Ilie Ser Ser Asp Phe 1 5 10 Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro 25 Val Thr Val Ala Ser Asn Leu Gin Asp Giu Glu Leu Cys Gly Gly Leu 40 Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val 55 Ala Gly Ser Lys Met Gin Gly Leu Leu Giu Arg Val Asn Thr Giu Ile WO 98/46750 PCT/US98/07511 His Phe Leu Leu Gly 145 Pro Cys Asn Leu Ala 225 Asn Pro Thr Val Gin Ala 115 Leu Gly Pro Ile Leu 195 Glu Lys Gln Ile Tyr 275 Lys Asn Lys Cys Pro Ser 165 Ile Asp Asn Leu Cys 245 Lys Val 70 Cys Ile Pro Gin Gly 150 Lys Asp Pro Leu Glu 230 Leu Ala Thr Ala Ser Trp Pro 135 Glu Glu Glu Asn Arg 215 Asn Pro Gly Leu Phe Arg Ile 120 Asp Pro Ser Ile Asn 200 Thr Ala Ser Asp Glu 280 Gin Leu 105 Thr Ser Ser His Ile 185 Leu Pro Ser Ala Trp 265 Gin Pro Leu Arg Ser Gly Lys 170 His Asn Asn Gly Thr 250 Gin Ala Pro Gin Gin Thr Pro 155 Ser His Ser Leu Ile 235 Ala Glu Gin Pro Glu Asn Leu 140 Ile Pro Leu Glu Leu 220 Glu Ala Phe Glu Ser Thr Phe 125 Tyr Ser Asn Lys Asp 205 Ala Ala Pro Arg Gin 285 Cys Ser 110 Ser Val Thr Met Arg 190 Met Phe Ile Ser Glu 270 Gin Arg Gin Cys Gly Asn 160 Asn Pro Ile Arg Arg 240 His Leu INFORMATION FOR SEQ ID NO:46: SEQUENCE CHARACTERISTICS: LENGTH: 313 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None Ala 1 Ala Val Trp Ala His Phe Leu Leu (xi) S Thr Gin Val Lys Thr Val Arg Leu Gly Ser Phe Val Val Gin Val Ala 115 Glu Leu 130

EQUENCE

Asp Cys 5 Ile Arg Ala Ser Val Leu Lys Met Thr Lys Thr Asn 100 Leu Lys Gin Cys DESCRIPTION: SEQ ID Ser Phe Gin His Ser Glu Leu Ser Asp Tyr Asn Leu Gin Asp Glu Ala Gin Arg Trp Met 55 Gin Gly Leu Leu Glu 70 Cys Ala Phe Gin Pro Ile Ser Arg Leu Leu 105 Pro Trp Ile Thr Arg 120 Gin Pro Asp Ser Ser 135 NO:46: Pro Ile Leu Leu Glu Leu Glu Arg Arg Val Pro Pro Gin Glu Gin Asn Thr Leu 140 WO 98/46750 Gly 145 Ser Arg His Gly Gly 225 Leu Val Pro Leu Val 305 Gly Ala Sei Leu 195 Trp Val Pro Leu Gin 275 Phe Gly Ser Pro His 180 Pro Lys Thr Thr Leu 260 Gly Gin Ser Pro Pro 165 Val Thr Thr Leu Cys 245 Leu Arg His Thr Gly Cys His Val Met 215 Leu Ser Ala Thr Leu 295 Cys Gly Asp Ser Leu 200 Giu G iu Ser Leu Ala 280 Arg Val 93 Ser Leu Arg 185 Leu Giu Gly Leu Gin 265 His Gly Arg PCT/US98/0751 I Gly Arg 170 Leu Pro Thr Val Leu 250 Ser Lys Lys Gly Leu Gin Val Ala 220 Ala Gin Leu Pro Arg 300 Asn Lys Pro 190 Phe Asp Arg Ser Thr 270 Al a Leu Met Leu 175 Glu Ser Ile Gly Gly 255 Gin Ile Met Ala 160 Leu Val1 Leu Leu Gin 240 Gin Leu Phe Leu INFORMATION FOR SEQ ID NO:47: SEQUENCE CHARACTERISTICS: LENGTH: 332 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu

I

Leu Val Leu Leu Gin Gin Leu Phe Leu 145 Giu Ile Arg His Gly Gly Leu Val Pro Leu 130 Val Gly Asn Asp Pro Giu Ala Gly Arg Pro 115 S er Gly Gly Pro Ser Leu Trp Val Pro Leu 100 Gin Phe Gly Gly Ser 5 His Pro Lys Thr Thr Leu Gly Gin Ser Gly 165 Pro Val Thr Thr Leu 70 Cys Leu Arg His Thr 150 Ser Pro His Val1 40 Met Leu Ser Ala Thr 120 Leu Cys Gly Lys Ser Leu Giu Glu Ser Leu 105 Ala Arg Val Glu Giu Arg Leu Glu Gly Leu 90 Gin His Gly Arg Pro 170 Ser Arg Leu Pro Thr Val 75 Leu Ser Lys Lys Glu 155 Ser His Val1 Ser Ala Lys Met Gly Leu Asp Val1 140 Phe Gly Lys Leu Gin Val Ala Ala Gin Leu Pro 125 Arg His Pro Ser Lys Pro Phe Asp Arg Ser Thr Ala Leu Tyr Ser 175 Asn Leu Glu Ser Ile Gly Gly Gin Ile Met Val 160 Thr Met WO 98/4675 Ala Ala Val 225 Trp Ala His Phe Leu 305 Leu 0o9 180 185 r Gin Asp Cys Ser Phe Gin His 195 200 1 Lys Ile Arg Giu Leu Ser Asp 0 215 r Val Ala Ser Asn Leu Gin Asp 230 g Leu Val Leu Ala Gin Arg Trp 245 y Ser Lys Met Gin Gly Leu Leu 260 265 e Val Thr Lys Cys Ala Phe Gin 275 280 1 Gin Thr Asn Ile Ser Arg Leu 0 295 1 Ala Leu Lys Pro Trp Ile Thr 310 u Leu Gin Cys Gin Pro Asp Ser 325 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 340 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear PCT/US98/0751 1 Ser Pro Tyr Leu Giu Giu 235 Met Giu 250 Giu Arg Pro Pro Leu Gin Arg Gin 315 Ser Thr 330 NO :48: Ile Leu 220 Leu Arg Val Pro Glu 300 Asn Leu Ser 205 Gin Cys Leu Asn Ser 285 Thr Phe Asp Tyr Gly Thr 255 Glu Leu Giu Arg Phe Pro Leu 240 Val Ile Arg Gin Cys 320 (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 48: Ala Thr Gin Asp Cys Ser Phe Gin His Ser i Ala Val Trp, Ala His Phe Leu Leu Gly 145 Pro Pro Val Thr Arg Gly Phe Val Val Giu 130 Gly Ser Arg Lys Val Leu Ser Val Gin Ala 115 Leu Gly Pro Leu Ile Ala Val Lys Thr Thr 100 Leu Gin Ser Pro Ile 180 Arg Ser Leu Met Lys Asn Lys Cys Pro Ser 165 Cys Giu Asn Ala Gin 70 Cys Ile Pro Gin Gly 150 Lys Asp Leu Leu Gin 55 Giy Ala Ser Trp Pro 135 Glu Glu Ser Ser Asp 25 Gin Asp 40 Arg Trp Leu Leu Phe Gin Arg Leu 105 Ile Thr 120 Asp Ser Pro Ser Ser His Arg Val 185 Pro Leu Giu Giu Arg 75 Pro Gin Gin Thr Pro 155 Ser Glu Ile Leu Leu Arg Val1 Pro Giu Asn Leu 140 Ile Pro Arg Ser Gin Cys Leu Asn Ser Thr Phe 125 Tyr Ser Asn Tyr Ser Asp Gly Lys Thr Cys Ser 110 Ser Vai Thr Met Leu 190 Phe Pro Leu Vai Ile Arg Gin Cys Giy Asn 160 Pro Glu Ala Lys Glu Aia Giu Asn Ile Thr Thr Gly Cys Ala Giu His Cys Ser WO 98/46750 95 PCT/US98/07511 Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala 210 215 220 Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp Gin Gly 225 230 235 240 Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu Leu Val 245 250 255 Asn Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp Lys Ala 260 265 270 Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala 275 280 285 Gin Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu 290 295 300 Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser 305 310 315 320 Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg 325 330 335 Thr Gly Asp Arg 340 INFORMATION FOR SEQ ID NO:49: SEQUENCE CHARACTERISTICS: LENGTH: 325 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49: Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu 1 5 10 Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His 25 Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe 40 Tyr Ala Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp 55 Gin Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu 70 75 Leu Val Asn Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp 90 Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu 100 105 110 Arg Ala Gin Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala 115 120 125 Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val 130 135 140 Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala 145 150 155 160 Cys Arg Thr Gly Asp Arg Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly 165 170 175 Gly Gly Ser Gly Gly Gly Ser Asn Met Ala Thr Gin Asp Cys Ser Phe 180 185 190 Gin His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu 195 200 205 Ser Asp Tyr Leu Leu Gin Asp Tyr Pro Val Thr Val Ala Ser Asn Leu WO 98/46750 WO 986750PT/US98/0751 1 Gin 225 Arg Leu Phe Arg Ile 305 Asp 21 As Tr Le Gi Le 29 Th Se 0 215 p Glu Giu Leu Cys Gly Gly Leu Trp Ar 230 23 p Met Glu Arg Leu Lys Thr Val Ala Gi 245 250 u Glu Arg Vai Asn Thr Giu Ile His Ph 260 265 n Pro Pro Pro Ser Cys Leu Arg Phe Va 275 280 u Leu Gin Glu Thr Ser Giu Gin Leu Va 0 295 r Arg Gin Asn Phe Ser Arg Cys Leu Gi 310 31 Ser Thr Leu 325 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1032 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear g 5 y e 1 5 220 Leu Ser Val Gin Ala 300 Leu Leu Met Lys 270 Asn Lys Cys Ala Gin 255 Cys Ile Pro Gin Gin 240 Gly Ala Ser Trp Pro 320 (xi)

GCTACACCAT

CAAGTGAGAA

120

AAGCTGTGCC

180

CCCCTGAGCT

240

AGCGGCCTTT

300

GGTCCCACCT

360

CAGATGGAAG

420

TTCGCCTCTG

480

TTCCTGGAGG

540

GGAGGCTCCC

600

AAAGAATCTC

660

ATCTCCTCCG

720

CCAGTCACCG

780

GTCCTGGCAC

840

TTGCTGGAGC

900 SEQUENCE DESCRIPTION: SEQ ID TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG

AGATCCAGGG

ACCCCGAGGA

CCTGCCCCAG

TCCTCTACCA

TGGACACACT

AACTGGGAAT

CTTTCCAGCG

TGTCGTACCG

CGGGTGAACC

ATAAATCTCC

ACTTCGCTGT

TGGCCTCCAA

AGCGCTGGAT

GCGTGAACAC

CGATGGCGCA

GCTGGTGCTG

CCAGGCCCTG

GGGGCTCCTG

GCAGCTGGAC

GGCCCCTGCC

CCGGGCAGGA

CGTTCTACGC

GTCTGGTCCA

AAACATGGCC

CAAAATCCGT

CCTGCAGGAC

GGAGCGGCTC

GGAGATACAC

GCGCTCCAGG

CTCGGACACT

CAGCTGGCAG

CAGGCCCTGG

GTCGCCGACT

CTGCAGCCCA

GGGGTCCTGG

CACCTTGCGC

ATCTCTACTA

ACCCAGGACT

GAGCTGTCTG

GAGGAGCTCT

AAGACTGTCG

TTTGTCACCA

AGAAGCTGTG

CTCTGGGCAT

GCTGCTTGAG

AAGGGATATC

TTGCCACCAC

CCCAGGGTGC

TTGCTAGCCA

AGCCCTACGT

TCAACCCGTC

GCTCCTTCCA

ACTACCTGCT

GCGGGGGCCT

CTGGGTCCAA

AATGTGCCTT

TGCCACCTAC

CCCCTGGGCT

CCAACTCCAT

CCCCGAGTTG

CATCTGGCAG

CATGCCGGCC

TCTGCAGAGC

AGAGGGCGGT

TCCTCCGTCT

ACACAGCCCC

TCAAGATTAC

CTGGCGGCTG

GATGCAAGGC

TCAGCCCCCC

WO 98/46750 WO 9846750PCT/US98/0751 I CCCAGCTGTC TTCGCTTCGT CCAGACCAAC ATCTCCCGCC TCCTGCAGGA GACCTCCGAG 960 CAGCTGGTGG CGCTGAAGCC CTGGATCACT CGCCAGAACT TCTCCCGGTG CCTGGAGCTG 1020 CAGTGTCAGC 'CC 1032 INFORMATION FOR SEQ ID NO:51: SEQUENCE CHARACTERISTICS: LENGTH: 1005 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

ATGGCCACTC

ATCCGTGAGC

120

CAGGACGAGG

180

CGGCTCAAGA

240

ATACACTTTG

300

ACCAACATCT

360

ATCACTCGCC

420

CTGTACGTAG

480

GCTACACCAT

540

CAAGTGAGAA

600

AAGCTGTGCC

660

CCCCTGAGCT

720

AGCGGCCTTT

780

GGTCCCACCT

840

CAGATGGAAG

900

TTCGCCTCTG

960

TTCCTGGAGG

1005 SEQUENCE DESCRIPTION: SEQ ID NO:51: AGGACTGCTC TTTTCAACAC AGCCCCATCT CCTCCGACTT CGCTGTCAAA

TGTCTGACTA

AGCTCTGCGG

CTGTCGCTGG

TCACCAAATG

CCCGCCTCCT

AGAACTTCTC

AGGGCGGTGG

TGGGCCCTGC

AGATCCAGGG

ACCCCGAGGA

CCTGCCCCAG

TCCTCTACCA

TGGACACACT

AACTGGGAAT

CTTTCCAGCG

TGTCGTACCG

CCTGCTTCAA

GGGCCTCTGG

GTCCAAGATG

TGCCTTTCAG

GCAGGAGACC

CCGGTGCCTG

AGGCTCCCCG

CAGCTCCCTG

CGATGGCGCA

GCTGGTGCTG

CCAGGCCCTG

GGGGCTCCTG

GCAGCTGGAC

GGCCCCTGCC

CCGGGCAGGA

CGTTCTACGC

GATTACCCAG

CGGCTGGTCC

CAAGGCTTGC

CCCCCCCCCA

TCCGAGCAGC

GAGCTGCAGT

GGTGGTGGTT

CCCCAGAGCT

GCGC!TCCAGG

CTCGGACACT

CAGCTGGCAG

CAGGCCCTGG

GTCGCCGACT

CTGCAGCCCA

GGGGTCCTGG

CACCTTGCGC

TCACCGTGGC

TGGCACAGCG

TGGAGCGCGT

GCTGTCTTCG

TGGTGGCGCT

GTCAGCCCGA

CTGGCGGCGG

TCCTGCTCAA

AGAAGCTGTG

CTCTGGGCAT

GCTGCTTGAG

AAGGGATATC

TTGCCACCAC

CCCAGGGTGC

TTGCTAGCCA

AGCCG

CTCCAACCTG

CTGGATGGAG

GAACACGGAG

CTTCGTCCAG

GAAGCCCTGG

CTCCTCAACC

CTCCAACATG

GTCTTTAGAG;

TGCCACCTAC

CCCCTGGGCT

CCAACTCCAT

CCCCGAGTTG

CATCTGGCAG

CATGCCGGCC

TCTGCAGAGC

INFORMATION FOR SEQ ID NO:52: SEQUENCE CHARACTERISTICS: LENGTH: 420 base pairs TYPE: nucleic acid WO 98/46750PCUS801 PC-r/US98/07511 STRANDEDNESS: single TOPOLOGY: linear (xi) 'SEQUENCE DESCRIPTION: GCCACTCAGG ACTGCTCTTT TCAACACAGC CGTGAGCTGT CTGACTACCT GCTTCAAGAT GACGAGGAGC TCTGCGGGGG CCTCTGGCGG

I

180

CTCAAGACTG

240

CACTTTGTCA

300

AACATCTCCC

360

ACTCGCCAGA

420

TCGCTGGGTC

CCAAATGTGC

GCCTCCTGCA

ACTTCTCCCG

SEQ ID NO:52: CCCATCTCCT CCGACTTCGC TACCCAGTCA CCGTGGCCTC CTGGTCCTGG CACAGCGCTG GGCTTGCTGG AGCGCGTGAA CCCCCCAGCT GTCTTCGCTT GAGCAGCTGG TGGCGCTGAA CTGCAGTGTC AGCCCGACTC

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

TGTCAAAATC

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCCTGGATC

CTCAACCCTG

INFORMATION FOR SEQ ID NO:53: SEQUENCE CHARACTERISTICS: LENGTH: 405 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCCACCCAGG

CGTGAGCTGT

120

GACGAGGAGC

180

CTCAAGACTG

240

CACTTTGTCA

300

AACATCTCCC

360

ACTCGCCAGA

405 SEQUENCE DESCRIPTION: ACTGCTCCTT CCAACACAGC

CTGACTACCT

TCTGCGGGGC

TCGCTGGGTC

CCAAATGTGC

GCCTCCTGCA

ACTTCTCCCG

GCTTCAAGAT

GCTCTGGCGG

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

SEQ ID NO:53: CCCATCTCCT CCGACTTCGC TACCCAGTCA CCGTGGCCTC CTGGTCCTGG CACAGCGCTG GGCTTGCTGG AGCGCGTGAA CCCCCCAGCT GTCTTCGCTT

TGTCAAAATC

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCCTGGATC! GAGCAGCTGG

CTGCAGTGTC

TGGCGCTGAA

AGCCC

INFORMATION FOR SEQ ID NO:54: SEQUENCE CHARACTERISTICS: LENGTH: 420 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54: WO 98/46750 GCCACCCAGG ACTGCTCCTT CGTGAGCTGT CTGACTACCT 120 GACGAGGAGC 'TCTGCGGGGG 180 CTCAAGACTG TCGCTGGGTC 240 CACTTTGTCA CCAAATGTGC 300 AACATCTCCC GCCTCCTGCA 360 ACTCGCCAGA ACTTCTCCCG 420

CCAACACAGC

GCTTCAAGAT

CCTCTGGCGG

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

CCCATCTCCT

TACCCAGTCA

CTGGTCCTGG

GGCTTGCTGG

CCCCCCAGCT

GAGCAGCTGG

CTGCAGTGTC

CCGACTTCGC

CCGTGGCCTC

CACAGCGCTG

AGCGCGTGAA

GTCTTCGCTT

TGGCGCTGAA

AGCCCGACTC

PCT/US98/0751 1

TGTCAAAATC

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCCTGGATC

CTCAACCCTG

INFORM4ATION FOR SEQ, ID SEQUENCE CHARACTERISTICS: LENGTH: 420 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCCACTCAGG

CGTGAGCTGT

120

GACGAGGAGC

180

CTCAAGACTG

240

CACTTTGTCA

300

AACATCTCCC

360

ACTCGCCAGA

420 SEQUENCE DESCRIPTION:

ACTGCTCTTT

CTGACTACCT

TCTGCGGGGG

TCGCTGGGTC

CCAAATGTGC

GCCTCCTGCA

ACTTCTCCCG

TCAACACAGC

GCTTCAAGAT

CCTCTGGCGG

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

SEQ ID CCCATCTCCT CCGACTTCGC TACCCAGTCA CCGTGGCCTC CTGGTCCTGG CACAGCGCTG GGCTTGCTGG AGCGCGTGAA CCCCCCAGCT GTCTTCGCTT GAGCAGCTGG TGGCGCTGAA CTGCAGTGTC AGCCCGACTC

TGTCAAAATC

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCCTGGATC

CTCAACCCTG

INFORMATION FOR SEQ ID NO:56: SEQUENCE CHARACTERISTICS: LENGTH: 942 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56: GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120 GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180 WO 98/46750 CTCAAGACTG TCGCTGGGTC 240 CACTTTGTCA CCAAATGTGC 300

AACATCTCCC'GCCTCCTGCA

360 ACTCGCCAGA ACTTCTCCCG 420

TACGTAGAGG

480

CCGTCTCCTC

540

TTCCAACACA

600

CTGCTTCAAG

660

GGCCTCTGGC

720

TCCAAGATGC

780

GCCTTTCAGC

840

CAGGAGACCT

900

CGGTGCCTGG

942

GCGGTGGAGG

CGTCTAAAGA

GCCCCATCTC

ATTACCCAGT

GGCTGGTCCT

AAGGCTTGCT

CCCCCCCCAG

CCGAGCAGCT

AGCTGCAGTG

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

CTCCCCGGGT

ATCTCATAAA

CTCCGACTTC

CACCGTGGCC

GGCACAGCGC

GGAGCGCGTG

CTGTCTTCGC

GGTGGCGCTG

TCAGCCCGAC

GGCTTGCTGG

CCCCCCAGCT

GAGCAGCTGG

CTGCAGTGTC

GAACCGTCTG

TCTCCAAACA

GCTGTCA.AAA

TCCAACCTGC

TGGATGGAGC

AACACGGAGA

TTCGTCCAGA

AAGCCCTGGA

TCCTCAACCC

AGCGCGTGAA

GTCTTCGCTT

TGGCGCTGAA

AGCCCGACTC

GTC!CAATCTC

TGGCTACCCA

TCCGTGAGCT

AGGACGAGGA

GGCTCAAGAC

TACACTTTGT

CCAACATCTC

TCACTCGCCA

TG

PCTIUS98/07511I

CACGGAGATA

CGTCCAGACC

GCCCTGGATC

CTCAACCCTG

TACTATCAAC

GGACTGCTCC

GTCTGACTAC

GCTCTGCGGG

TGTCGCTGGG

CACCAAATGT

CCGCCTCCTG

GAACTTCTCC

INFORMATION FOR SEQ ID NO:57: SEQUENCE CHARACTERISTICS: LENGTH: 1003 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: GGCCACTCAG GACTGCTCTT TTCAACACAG

CCGTGAGCTG

120

GGACGAGGAG

180

GCTCAAGACT

240

ACACTTTGTC

300

CAACATCTCC

360

CACTCGCCAG

420

GTACGTAGAG

480

TACACCATTG

540

AGTGAGAAAG

600

TCTGACTACC

CTCTGCGGGG

GTCGCTGGGT

ACCAAATGTG

CGCCTCCTGC

AACTTCTCCC

GGCGGTGGAG

GGCCCTGCCA

ATCCAGGGCG

TGCTTCAAGA

GCCTCTGGCG

CCAAGATGCA

CCTTTCAGCC

AGGAGACCTC

GGTGCCTGGA

GCTCCCCGGG

GCTCCCTGCC

ATGGCGCAGC

SEQ ID NO:57: CCCCATCTCC TCCGACTTCG TTACCCAGTC ACCGTGGCCT GCTGGTC!CTG GCACAGCGCT AGGCTTGCTG GAGCGCGTGA CCCCCCCAGC TGTCTTCGCT CGAGCAGCTG GTGGCGCTGA GCTGCAGTGT CAGCCCGACT TGGTGGTTCT GGCGGCGGCT CCAGAGCTTC CTGCTCAAGT GCTCCAGGAG AAGCTGTGTG

CTGTCAAAAT

CCAACCTGCA

GGATGGAGCG

ACACGGAGAT

TCGTCCAGAC

AGCCCTGGAT

CCTCAACCCT

CCAACATGGC

CTTTAGAGCA

CCACCTACAA

WO 98/46750 GCTGTGCCAC CCCGAGGAGC 660 CCTGAGCTCC TGCCCCAGCC 720 CGGCCTTTTC' CTCTACCAGG 780 TCCCACCTTG GACACACTGC 840 GATGGAAGAA CTGGGAATGG 900 CGCCTCTGCT TTCCAGCGCC 960 CCTGGAGGTG TCGTACCGCG 1003

TGGTGCTGCT

AGGCCCTGCA

GGCTCCTGCA

AGCTGGACGT

CCCCTGCCCT

GGGCAGGAGG

TTCTACGCCA

CGGACACTCT

GCTGGCAGGC

GGCCCTGGAA

CGCCGACTTT

GCAGCCCACC

GGTCCTGGTT

CCTTGCGCAG

CTGGGCATCC

TGCTTGAGCC

GGGATATCCC

GCCACCACCA

CAGGGTGCCA

GCTAGCCATC

CCG

PCT/US98/0751 I

CCTGGGCTCC

AACTCCATAG

CCGAGTTGGG

TCTGGCAGCA

TGCCGGCCTT

TGCAGAGCTT

INFORMATION FOR SEQ ID NO:58: SEQUENCE CHARACTERISTICS: LENGTH: 858 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi)

GCCACCCAGG

CGTGAGCTGT

120

GACGAGGAGC

180

CTCAAGACTG

240

CACTTTGTCA

300

AACATCTCCC

360

ACTCGCCAGA

420

TACGTAGAGG

480

CCGTCTCCTC

540

ATCGATGAAA

600

CTCAATTCTG

660

GCATTCGTAA

720

AATCTCCAAC

780

AAGGCAGGTG

840

CAAGCGCAGG

SEQUENCE DESCRIPTION: SEQ ID NO:58: ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC

CTGACTACCT

TCTGCGGGGG

TCGCTGGGTC

CCAAATGTGC

GCCTCCTGCA

ACTTCTCCCG

GCGGTGGAGG

CGTCTAAAGA

TTATACATCA

AAGACATGGA

GGGCTGTCAA

CATGTCTGCC

ACTGGCAAGA

AACAACAG

GCTTCAAGAT

CCTCTGGCGG

CAAGATGCAA

CTTTCAGCCC

GGAGACCTCC

GTGCCTGGAG

CTCCCCGGGT

ATCTCATAAA

CTTAAAGAGA

TATCCTGATG

GCACTTAGAA

CTCTGCCACG

ATTCCGGGAA

TACCCAGTCA

CTGGTCCTGG

GGCTTGCTGG

CCCCCCAGCT

GAGCAGCTGG

CTGCAGTGTC

GAACCGTCTG

TCTCCAAACA

CCACCTAACC

GAACGAAACC

AATGCATCAG

GCCGCACCCT

AAACTGACGT

CCGTGGCCTC

CACAGCGCTG

AGCGCGTGAA.

GTCTTCGCTT

TGGCGCTGAA

AGCCCGACTC

GTCCAATCTC

TGGCTAACTG

CTTTGCTGGA

TTCGAACTCC

GTATTGAGGC

CTCGACATCC

TCTATCTGGT

CAACCTGCAG

GATGGAGCGG

CACGGAGATA

CGTCCAGACC

GCCCTGGATC

CTCAACCCTG

TACTATCAAC

CTCTATAATG

CCCGAACAAC

AAACCTGCTC

AATTCTTCGT

AATCATCATC

TACCCTTGAG

INFORMATION FOR SEQ ID N0:59: WO 98/46750 PCT/US98/07511 SEQUENCE CHARACTERISTICS: LENGTH: 402 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59: ATGGCTCCAA TGACTCAGAC TACTTCTCTT AAGACTTCTT GGGTTAACTG ATCGATGAAA TTATAACACA CTTAAAGCAG CCACCTTTGC CTTTGCTGGA 120 CTCAATGGGG AAGACCAAGA CATTCTGATG GAAAATAACC TTCGAAGGCC 180 GCATTCAACA GGGCTGTCAA GAGTTTACAG AATGCATCAG CAATTGAGAG 240 AATCTCCTGC CATGTCTGCC CCTGGCCACG GCCGCACCCA CGCGACATCC 300 AAGGACGGTG ACTGGAATGA ATTCCGTCGT AAACTGACCT TCTATCTGAA 360 AACGCGCAGG CTCAACAGAC CACTCTGTCG CTAGCGATCT TT 402

CTCTAACATG

CTTCAACAAC

AAACCTGGAG

CATTCTTAAA

AATCCATATC

AACCTTGGAG

INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 344 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID Ala Thr Pro Leu Gly 1 Lys Gin Val Cys Ser Ser Asp Pro Phe 145 Ser Glu Leu Pro Gly Pro Phe Ala 130 Gin Leu Lys Leu Ser Leu Glu Ala 115 Leu Arg Glu Leu Gly Gin Phe Leu 100 Thr Gin Arg 5 Gin Cys His Ala Leu Gly Thr Pro Ala Pro Val Ala Ser Leu 70 Tyr Pro Ile Thr Gly 150 Ala Arg Thr Leu 55 Gin Gin Thr Trp Gin 135 Gly Ser Lys Tyr 40 Gly Leu Gly Leu Gin 120 Gly Val Ser Ile 25 Lys Ile Ala Leu Asp 105 Gin Ala Leu Leu 10 Gin Leu Pro Gly Leu 90 Thr Met Met Val Pro Gin Gly Asp Cys His Trp Ala Cys Leu 75 Gin Ala Leu Gin Glu Glu Pro Ala 140 Ala Ser 155 Ser Gly Pro Pro Ser Leu Leu Leu 125 Phe His Phe Ala Glu Leu Gin Glu Asp 110 Gly Ala Leu Leu Ala Glu Ser Leu Gly Val Met Ser Gin Leu Leu Leu Ser His Ile Ala Ala Ala Ser 160 WO 98/46750 PCT/US98/07511 Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gin Pro Tyr 165 170 175 Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser 180 185 190 Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn 195 200 205 Met Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro Ile Ser Ser Asp 210 215 220 Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr 225 230 235 240 Pro Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly 245 250 255 Leu Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr 260 265 270 Val Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu 275 280 285 Ile His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu 290 295 300 Arg Phe Val Gin Thr Asn Ile Ser Arg Leu Leu Gin Glu Thr Ser Glu 305 310 315 320 Gin Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gin Asn Phe Ser Arg 325 330 335 Cys Leu Glu Leu Gin Cys Gin Pro 340 INFORMATION FOR SEQ ID NO:61: SEQUENCE CHARACTERISTICS: LENGTH: 133 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61: Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser 1 5 10 Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gin Pro Pro Leu Pro 25 Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp Ile Leu Met 40 Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val 55 Lys Ser Leu Gin Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu 70 75 Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile 90 His Ile Lys Asp Gly Asp Trp Asn Gly Ile Phe Arg Arg Lys Leu Thr 100 105 110 Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gin Ala Gin Gin Thr Thr Leu 115 120 125 Ser Leu Ala Ile Phe 130 INFORMATION FOR SEQ ID NO:62: WO 98/46750 WO 9846750PCTIUS98/0751 1 SEQUENCE CHARACTERISTICS: LENGTH: 287 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62: Pro Met Thr Gin Thr Thr Ser Leu Lys Thr 1 Asn Leu Glu Lys Leu His Phe Ser Gly 145 His Asp Asp Trp, Leu 225 Gin Leu Thr Met Leu Asn Ser Pro Ile Tyr Leu 130 Ser Ser Tyr Giu Met 210 Giu Pro Leu Arg Ile Asp Asp Phe Asn Leu Leu Gin Cys Leu Lys Asp 100 Leu Lys 115 Ala Ile Gly Gly Pro Ile Leu Leu 180 Giu Leu i9 5 Giu Arg Arg Val Pro Pro Gin Giu 260 Gin Asn 5 Glu Asn Arg Asn Pro Gly Thr Phe Gly Ser 165 Gin Cys Leu Asn Ser 245 Thr Phe Ile Asn Arg Ala 70 Leu Asp Leu Tyr Ser 150 Ser Asp Gly Lys Thr 230 Cys Ser Ser Ile Leu Pro 55 Ser Ala Trp Glu Val 135 Asn Asp Tyr Ala Thr 215 Giu Leu Glu Arg Thr Asn 40 Asn.

Al a Thr Asn Asn 120 Giu Met Phe Pro Leu 200 Val Ile Arg Gin Cys 280 His Gly Leu Ile Ala Gly 105 Ala Gly Ala Ala Val 185 Trp Ala His Phe Leu 265 Leu 10 Leu Glu Glu Glu Al a 90 Ile Gin Gly Thr Val 170 Thr Arg Gly Phe Val 250 Val Glu Ser Lys Asp Ala Ser 75 Pro Phe Ala Gly Gin 155 Lys Val Leu Ser Val 235 Gin Ala Leu Trp Gin Gin Phe Ile Thr Arg Gin Gly 140 Asp Ile Ala Val Lys 220 Thr Thr Leu Gin.

Val Pro Asp Asn Leu Arg Arg Gin 125 Ser Cys Arg Ser Leu 205 Met Lys Asn Lys Cys 285 Cys Leu Leu Ala Asn Pro Leu Thr Gly Phe Leu 175 Leu Gin Gly Ala Ser 255 Trp, Pro Ser Pro Met Val Leu Ile Thr Leu Gly Gin 160 Ser Gin Arg Leu Phe 240 Arg Ile INFORMATION FOR SEQ ID NO:63: SEQUENCE CHARACTERISTICS: LENGTH: 302 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63: WO 98/46750 PCT/US98/07511 Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser 1 5 10 Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gin Pro Pro Leu Pro 1 20 25 Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp Ile Leu Met 40 Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val 55 Lys Ser Leu Gin Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu 70 75 Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile 90 His Ile Lys Asp Gly Asp Trp Asn Gly Ile Phe Arg Arg Lys Leu Thr 100 105 110 Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gin Ala Gin Gin Thr Thr Leu 115 120 125 Ser Leu Ala Ile Phe Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu 130 135 140 Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu 145 150 155 160 Ser His Lys Ser Pro Asn Met Ala Thr Gin Asp Cys Ser Phe Gin His 165 170 175 Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp 180 185 190 Tyr Leu Leu Gin Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gin Asp 195 200 205 Glu Glu Leu Cys Gly Ala Leu Trp Arg Leu Val Leu Ala Gin Arg Trp 210 215 220 Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gin Gly Leu Leu 225 230 235 240 Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gin 245 250 255 Pro Pro Pro Ser Cys Leu Arg Phe Val Gin Thr Asn Ile Ser Arg Leu 260 265 270 Leu Gin Glu Thr Ser Glu Gin Leu Val Ala Leu Lys Pro Trp Ile Thr 275 280 285 Arg Gin Asn Phe Ser Arg Cys Leu Glu Leu Gin Cys Gin Pro 290 295 300 INFORMATION FOR SEQ ID NO:64: SEQUENCE CHARACTERISTICS: LENGTH: 407 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64: CCATGGCTAA CTGCTCTATA ATGATCGATG AAATTATACA TCACTTAAAG AGACCACCTG CACCTTTGCT GGACCCGAAC AACCTCAATG ACGAAGACGT CTCTATCCTG ATGGATCGAA 120 ACCTTCGACT TCCAAACCTG GAGAGCTTCG TAAGGGCTGT CAAGAACTTA GAAAATGCAT 180 1 1 WO 98/46750 PCT/US98/07511 CAGGTATTGA GGCAATTCTT CGTAATCTCC AACCATGTCT GCCCTCTGCC ACGGCCGCAC 240 CCTCTCGACA TCCAATCATC ATCAAGGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA 300 CGTTCTATCT GGTTACCCTT GAGCAAGCGC AGGAACAACA GTACGTAGAG GGCGGTGGAG 360 GCTCCCCGGG TGGTGGTTCT GGCGGCGGCT CCAACATGTA AGGTACC 407 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 452 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID CCATGGCTAA CTGCTCTATA ATGATCGATG AAATTATACA TCACTTAAAG CACCTTTGCT GGACCCGAAC AACCTCAATG ACGAAGACGT CTCTATCCTG 120 ACCTTCGACT TCCAAACCTG GAGAGCTTCG TAAGGGCTGT CAAGAACTTA 180 CAGGTATTGA GGCAATTCTT CGTAATCTCC AACCATGTCT GCCCTCTGCC 240 CCTCTCGACA TCCAATCATC ATCAAGGCAG GTGACTGGCA AGAATTCCGG 300 CGTTCTATCT GGTTACCCTT GAGCAAGCGC AGGAACAACA GTACGTAGAG 360 GCTCCCCGGG TGAACCGTCT GGTCCAATCT CTACTATCAA CCCGTCTCCT 420 AATCTCATAA ATCTCCAAAC ATGTAAGGTA CC 452

AGACCACCTG

ATGGATCGAA

GAAAATGCAT

ACGGCCGCAC

GAAAAACTGA

GGCGGTGGAG

CCGTCTAAAG

Claims (8)

1. A chimeric protein comnprising a polypeptide, having a formula selected from the group consisting RJ-L-R 2 R2-L-RI, Rj-R2, R 2 -Rj, Met-Ala-R1-L- R2, Met-Ala-R2 -L-RI, Met-Ala-R1-R2, Met-Ala-R2-Rl, Ala-R1-L-R2, Ala-R2--L-RI, Ala-Rl-R2 and Ala-R2- Rj; wherein in R1 is a flt3 ligand; R2 is selected from the group consisting of G- CSF or G-CSF Serl 7 and L is a linvcer capable of linking R1 to R2. :20 2. The chimeric protein of claim 1 selected *from the group consisting of; th prti.ain*. euec fS-QI O9 ~the protein hiaving the sequence of SEQ ID NO:1; the protein having the sequence of- SEQ ID NO:ll; :the protein having the sequence of SEQ ID NO;38; the protein havinig the sequence of SEQ ID and the proteiLn having the gecluence of S] Q TO NO: 108

3. A pharmaceutical composition comprising a chimera protein of claim 1 or 2 and a pharmaceutically acceptable carrier.

4. A nucleic acid molecule encoding the chimera protein of claim 1. A nucleic acid molecule encoding the chimera protein of claim 2.

6. A nucleic acid molecule of claim selected from the group consisting of: a DNA sequencing having the sequence of SEQ ID NO:10; and a DNA sequencing having the sequence of SEQ ID NO:12; 20 a DNA sequencing having the sequence of SEQ ID NO;17; a DNA sequencing having the sequence of SEQ ID NO:19; a DNA sequencing having the sequence of SEQ ID and a DNA sequencing having the sequence of SEQ ID

7. A method of producing a chimera protein comprising: growing under suitable nutrient conditions, a host cell transformed or transfected with a replicable vector comprising said nucleic acid molecule of claim 4, 5, or 6 in a manner allowing expression of said chimera protein and recovering said chimera protein. 109

8. A method of increasing hematopoietic cell production in a mammal in need thereof comprising administering a pharmaceutically effective amount of the chimera protein of claims 1 or 2.

9. A method of increasing hematopoietic cell production in a mammal in need thereof comprising administering a pharmaceutically effective amount of the composition of claim 3. A method for ex vivo expansion of stem cells, comprising the steps of; culturing said stem cells with a selected growth medium comprising a chimeric protein of claims 1 or 2; and harvesting said cultured stem cells. 20 11. A method of increasing hematopoietic cell production in a mammal in need thereof comprising administering a pharmaceutically effective amount of the expanded stem cells of claim

12. A method of human gene therapy, comprising the steps of; removing stem cells from a patient or donor culturing said stem cells with a selected V 60 30 growth medium comprising a chimera protein of claim 1 or 2; transducing DNA into said cultured cells; harvesting said transduced cells; and transplanting said transduced cells into said patient. DATED this 4 day of July 2002 G. D. SEARLE CO., By its Patent Attorneys, SF. WELLINGTON CO., We elfington) "I uo