EP0973900A1 - flt3 LIGAND CHIMERIC PROTEINS - Google Patents

Abstract

Disclosed are novel chimeric proteins or multi-functional hematopoietic receptor agonist proteins comprising a flt3 agonist, DNAs which encode the multi-functional hematopoietic receptor agonists proteins, methods of making the multi-functional hematopoietic receptor agonists proteins and methods of using the multi-functional hematopoietic receptor agonists proteins.

Description

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 macrophage colonies, respectively while GM-CSF and interl,eu in-3 (I -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 treating patients with a hematopoietic disorder using flt3 ligand.

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 10 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 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 Tyr¹, Tyr³ 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 (F. 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 (K. 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 (i.e., 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 Cys⁷-to-His⁷ mutation eliminates biological activity. A series of mutant erythropoietins with a single amino acid substitution at asparagine residues 24, 38 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 (S. 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 vi tro bioassays (M. 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 (P. H..

Lowy et al . , Nature 185:102-105 (1960); E. Goldwasser and C. K. H.. Kung, Ann . N. Y. Acad. Science 149:49-53 (1968); W. A. Lukowsky and R. H.. 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 plasma half-life of glycosylated and deglycosylated erythropoietin (J.C. 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) (Y. Chern et al . , Eur. J. Biochem. 202:225-230 (1991)). The domain 1 mutants are rapidly degraded and inactive in an in vi tro bioassay while the domain 2 mutants, at best, retain in vi tro activity. These mutants also show no enhanced in vivo biological activity as compared to wild-type, human erythropoietin. These authors 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 (P.H. Lai et al . , J. Biol . Chem . 261:3116-3121 (1986)). Oligonucleotide- directed 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 vi tro 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 (F.K. Lin, Molecular and Cellular Aspects of Erythropoietin and Erythropoieεis, 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. 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 (Asn⁶⁹), EPO (As^'n¹²⁵, Ser¹²⁷), EPO (Thr¹²⁵), and EPO (Pro¹²⁴, Thr¹²⁵).

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 (X³³, Cys¹³⁹, des-Arg¹⁶⁶) and EPO

(Cys¹³⁹, des-Arg¹⁶⁶) .

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 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"148, SCF^1"157, SCF^1"160, SCF^1"161, SCF^1"162, SCF^1"164, SCF^1"165, SCF^1"183, SCF^1"185, SCF^1"

188 _{f S C F}1-189 _{; S CF}l-2 _{0 S CF}l- 48 _

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 Ser8 -> Pro8 replacement.

Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites. 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, 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 Cell- derived 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), 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-5. IL 6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-15, 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 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 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: (1) 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 (2) 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), IL-5 (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 α- chains become associated with at least one other receptor chain (β-chain, γ-chain) . Many of these factors share a common receptor subunit. The α-chains for GM-CSF, IL-3 and IL-5 share the same β-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 β-chain (gpl30) (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 γ- chain (Kondo et al . , Science 262:1874, 1993; Russell 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 side- effects.

Novel compounds of this invention are represented by a formula selected from the group consisting of:

R1-L1-R2, R2-L1-R1, R1-R2, and R2-R1

Where Ri 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 Ri . By complementary activity is meant activity which enhances or changes the response to another cell modulator. The Ri 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 Li represents a chemical bond or polypeptide segment to which both Ri and R2 are joined in frame, most commonly Li is a linear peptide to which Rl and R2 are joined by amide bonds linking the carboxy terminus of Ri to the amino terminus of Li and carboxy terminus of Li to the 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 Ri 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 Ri 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-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 IL- 12. Additionally, this invention encompasses the use of modified Ri or R2 molecules or mutated or modified DNA sequences encoding these Ri 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 (1) allow the two molecules to fold and act independently of each other, (2) not have a propensity for developing an ordered secondary structure which could interfere with the functional domains of the two proteins, (3) have minimal hydrophobic characteristics which could interact with the functional protein domains and (4) provide steric separation of Ri and R2 such that Ri 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 Li linkers of the present invention include sequences selected from the group of formulas:

(Gly³Ser)ⁿ (SEQ ID NO:l), (Gly⁴Ser)ⁿ (SEQ ID NO:2), (Gly⁵Ser)ⁿ (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 pill 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 pill surface protein . The spacer region consists of the amino acid sequence :

GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGluGlyGlyGlySerGlu GlyGlyGlySerGluGlyGlyGlySerGluGlyGlyGlySerGlyGlyGlySer ( SEQ ID NO : 6 ) . 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:

IleSerGluProSerGlyProIleSerThrlleAsnProSerProProSer ys GluSerHisLysSerPro (SEQ ID NO : 7 ) , and

IleGluGlyArglleSerGluProSerGlyProIleSerThrlleAsnProSer

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 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 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 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 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: 25- 33, 1993). Various strains of B. subtilis may also be employed in this method. Many strains of yeast cells known to those skilled in the art are also available as host cells for expression of the polypeptides of the present invention. When expressed in the E. coli cytoplasm, the gene encoding the 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

-2 -1 -1 encode Met -Ala - or Met at the N-termmus of the protein. The N termini of proteins made in the cytoplasm of E. coli are affected by post- translational processing by methionine aminopeptidase (Ben Bassat et al . , J. Bac . 169:751-757 , 1987) and possibly by other peptidases so that upon expression the methionine is cleaved off the N-terminus. The multi functional hematopoietic receptor agonists of the present invention may include multi-functional hematopoietic receptor agonist polypeptides having

-1 -1 -2 -1 Met , Ala or Met -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. J., 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 C0S7 , HeLa, BHK, CHO, or mouse L lines. The cells can be cultured, for example, in DMEM media (JRH Scientific) . The polypeptide secreted into the media can be recovered by standard biochemical approaches following transient expression for 24 - 72 hours after transfection of the cells or after establishment of stable cell lines following selection for antibiotic resistance. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art. See, e.g., Gething and Sambrook, Nature, 293:620-625, 1981), or alternatively, Kaufman et al, Mol . Cell . Biol . , 5 (7) : 1750-1759 , 1985) or Howley et al., U.S. Pat. No. 4,419,446. Another suitable mammalian cell line is the monkey COS-1 cell line. A similarly useful mammalian cell line is the CV-1 cell line.

Where desired, insect cells may be utilized as host cells in the method of the present invention. See, e.g., Miller et al . , Genetic Engineering, 8:277- 298 (Plenum Press 1986) and references cited therein. In addition, general methods for expression of foreign genes in insect cells using Baculovirus vectors are described in: Summers, M. D. and Smith, G. E., 1987) - A manual of methods for Baculovirus vectors and insect cell culture procedures, Texas Agricultural Experiment Station Bulletin No. 1555. An expression vector is constructed comprising a Baculovirus transfer vector, in which a strong Baculovirus promoter (such as the polyhedron promoter) drives transcription of a eukaryotic secretion signal peptide coding region, which is translationally 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 co- transfected with one microgram of Baculovirus DNA (see Summers & Smith, 1987) into insect cells, strain 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, e.g., 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- 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 allo- antibodies 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 cell collection and dramatically decreased the cost of the 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, co- administered with other CSFs, or in combination with bone marrow transplants subsequent to high dose chemotherapy to treat the neutropenia and thrombocytopenia which are often the result of such treatment. However the period of severe neutropenia and thrombocytopenia may not be totally eliminated. The myeloid lineage, which is comprised of monocytes (macrophages) , granulocytes (including neutrophils) and megakaryocytes, is critical in preventing infections and bleeding which can be life- threatening. Neutropenia and thrombocytopenia may also be the result of disease, genetic disorders, drugs, toxins, radiation and many therapeutic treatments such as conventional oncology therapy.

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

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

5,061,620 relates to compositions comprising human hematopoietic stem cells provided by separating the stem cells from dedicated cells . 5,199,942 describes a method for autologous hematopoietic cell transplantation comprising: (1) obtaining hematopoietic progenitor cells from a patient; (2) 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 yeloblasts 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: (a) separating stem cells from other cells, (b) culturing said separated stem cells with a selective media which contains multi-functional hematopoietic receptor agonist protein (s) and (c) 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-1+ and lin-, but it is to be understood that the present invention is not limited to the expansion of this stem cell population. The CD34+ enriched human stem cell fraction can be separated by a number of reported methods, including affinity columns or beads, magnetic beads or flow cytometry using antibodies directed to surface antigens such as the CD34+. Further, physical separation methods such as counterflow elutriation may be used to enrich hematopoietic progenitors. The CD34+ progenitors are heterogeneous, and may be divided into several sub- populations characterized by the presence or absence of co-expression of different lineage associated cell surface associated molecules. The most immature progenitor cells do not express any known lineage associated markers, such as HLA-DR or CD38, but they may express CD90(thy-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 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 (e.g. 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-1 (Muench et al . , Blood 81:3463-3473 [1993] ) IL-6 (Sato et al . , Blood 82:3600-3609 [1993] ) IL-11 (Lemoli et al . , Exp . Hem. 21:1668-1672 [1993], Sato et al . , Blood 82:3600-3609 [1993]), 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., Bio therapy 7 : 13 -26 [1994], Sato et al . , Blood 82:3600-3609 [1993], Smith et al . , Exp . Hem. 21:870- 877 [1993], Steen et al . , Stem Cells 12:214-224 [1994], Tsujino et al . , Exp . Hem. 21:1379-1386

[1993]). Among the individual colony stimulating factors, hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609 [1993], Kobayashi et al., Blood 73:1836-1841 [1989]). However, no single factor has been shown to be as effective as the combination of multiple factors . The present invention provides methods for ex vivo expansion that utilize 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 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, BioTechniqu.es 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 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, e.g., dialysis. The multi-functional hematopoietic receptor agonists of the present invention may be useful in treating such hematopoietic deficiencies.

Various immunodeficiencies, e.g., in T and/or B lymphocytes, or immune disorders, e.g., rheumatoid arthritis, may also be beneficially affected by treatment with the multi-functional hematopoietic receptor agonists of the present invention. Immunodeficiencies may be the result of viral infections, e.g., HTLVI, HTLVII, HTLVIII, severe exposure to radiation, cancer therapy or the result of other medical treatment. The 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. 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, e.g., the condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. Generally, a daily regimen may be in the range of 0.2 - 150 μg/kg of multi-functional hematopoietic receptor agonist protein per kilogram of body weight. 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 co- administration with the chimeric proteins of the present invention includes GM-CSF, G-CSF, G-CSF

Ser¹⁷, 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-10, 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 above would be adjusted to compensate for such additional components in the therapeutic composition. Progress of the treated patient can be monitored by periodic assessment of the hematological profile, e.g., differential cell count and the like.

Determination of 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 vi tro (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 IX PBS (#14040.059 Life Technologies, Gaithersburg, MD. ) solution in a 50 mL conical tube (#25339-50 Corning, Corning MD) . Ficoll (Histopaque 1077 Sigma H-8889) is layered under the diluted sample and centrifuged, 300 x g for 30 min. The mononuclear cell band is removed and washed two times in IX PBS and once with 1% BSA PBS (Cell ro 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 °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 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 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, BC . ) . 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, B., et al . , Blood 70: 192, 1987; Valtieri, M., 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

5 then replated at 1x10 cells/well a 24 well plate in media containing 100 U/mL IL-3. It took approximately 2 months for the cells to grow rapidly in IL-3. These cells are maintained as AML 193 1.3 thereafter by supplementing tissue culture medium (see below) with human IL-3.

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

5 5 a density ranging from 2 x 10 to 5 x 10 viable cells/mL. This medium is prepared by supplementing Iscove ' s modified Dulbecco ' s Medium (IMDM, Hazelton, Lenexa, KS) with albumin, transferrin, lipids and 2- mercaptoethanol . Bovine albumin (Boehringer-

Mannheim, Indianapolis, IN) is added at 500 μg/mL; human transferrin (Boehringer-Mannheim, Indianapolis, IN) is added at 100 μg/mL; soybean lipid (Boehringer- Mannheim, Indianapolis, IN) is added at 50 μg/mL; and 2-mercaptoethanol (Sigma, St. Louis, MO) is added at

5 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 μl 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

₄ 50 μl (2.5 x 10 cells) mto each well. Tissue culture plates are incubated at 37°C with 5% CO₂ in humidified air for 3 days. On day 3, 0.5 μCi ³H- thymidine (2 Ci/mM, New England Nuclear, Boston, MA) is added in 50 μl of tissue culture medium. Cultures are incubated at 37°C with 5% CO₂ 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 70% 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 ³H-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^' (³H-thymidine incorporation) induced by graded concentrations of interleukin-3 or multi- functional chimeric hematopoietic receptor agonist.

₅ Typically, concentration ranges from 0.05 pM - 10 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 measured by ³H-thymidine incorporation observed in triplicate cultures lacking interleukin-3 ) . This EC₅₀ value is also equivalent to 1 unit of bioactivity. Every assay 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. TFl c-mpl ligand dependent proliferation assay

The c-mpl ligand proliferative activity can be assayed using a subclone of the pluripotential human cell line TFl (Kitamura et al . , J. Cell Physiol

140:323-334. [1989]). TFl 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 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 penicillin- streptomycin (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, 50 ug/mL soybean lipids, 4 x 10-8M beta- mercaptoethanol 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 10 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 1.8%. Triplicate samples of a dose curve of IL-3 variant pMONl3288 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% CO₂ and 37° 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% CO₂ and 37° C for four hours. The plate is harvested and counted on a Betaplate counter.

MUTZ-2 Cell Proliferation Assay 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 (20- lOOng/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°C and 5%C0₂ 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. 50μl of assay media is loaded into all wells except row A. 75μl of the flt3 receptor agonists or standards are added to row A and 25μl taken from that row and serial dilutions (1:3) performed on the rest of the plate (rows B through G) . Row H remains as a media only control. The starved MUTZ-2 cells are washed two times in IMDM medium and resuspended in 50μl assay media. 50μl 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°C and 5%C0₂ for

44hrs . Each well is then pulsed with lμCi/well of tritiated thymidine in a volume of 20μl for four hours. Plates are then harvested and counted.

Other in vitro cell based proliferation assays 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.

TFl proliferation assay: TFl 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 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 Ca.). 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 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 μl of supernatant added to 1 mL cultures (approximately a 10% dilution) . Cells were incubated at 37°C for 8-14 days at 5% C0₂ in a 37°C humidified incubator.

b. Cell Harvest and Analysis:

At the end of the culture period a total cell count 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 μm PGE1, 11 mM glucose, 3% w/v BSA, in PBS, pH 7.4,) (Tomer et al., Blood 70: 1735-1742, 1987) resuspended in 500 μl 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 20 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 (Calbiochem , La Jolla Ca) (50 μg/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 (Beeton 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.

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, 6.67μM FeCl₂ , 25μg/mL

CaCl₂, 25μg/mL L-asparagine, 500μg/mL e-amino-n- caproic 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°C for 13 days at 5% C0₂ in a 37°C humidified incubator.

At the end of the culture period plates were fixed with methanol : acetone (1:3), air dried and stored at -200C until staining. A peroxidase immunocytochemistry staining procedure was used (Zymed, Histostain-SP. San Francisco, CA) using a 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. 25 cells), BFU-MK (large, multi-foci colonies with > 25 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 Ncol/Hindlll 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 Afllll/Hindlll and SAP-treated (pMON30311 is a BHK-specific vector containing NcoI-IL-3 receptor agonist-IgG2B-Afllll-Hindlll as an insert) using standard ligation conditions. The ligation mixtures were used to transform competent DH5a cells (Gibco BRL cat #18265-017) following the manufacturer's recommended protocol, and vector 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 Ncol/Afllll restriction fragment from pMON30244 (GlySer linker) SEQ ID NO: 65 and pMON30245 (IgG2B linker) SEQ ID NO: 66 respectively, into vector, pMON30237, digested with Ncol (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, 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 Ncol/Afllll restriction fragment from pMON30244 (GlySer linker) and pMON30245 (IgG2B linker) respectively, into vector, pMON30238, digested with Ncol (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 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 NO:30 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°C, one minute, 65°C, two minutes, and 72°C 2 1/2 minutes; followed by ten cycles of 94°C, one minute, 70°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 μl dH₂0. 20 μl of each purified PCR product were digested in 50 μl reaction mixture volumes with 10U each of Ncol and SnaBI for 90 minutes at 37°C. One μg of vector, PMON26431 (derivative of pMONl3061 - W095/21254), was digested with 7.5U each of Ncol and SnaBI in a 20 μl reaction volume for 90 minutes at 37°C, followed by the addition of 1U shrimp alkaline phosphatase. The reaction was incubated an additional 10 minutes at 37°C, and both inserts and vector were gel purified as described previously. Ligation times and temperatures were modified to include incubation at 16°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 Ncol/Hindlll restriction fragment from pMON30237 into plasmid, pMON30309 (derivative of pMONl3149 - W095/21254), digested with Afllll/Hindlll (contains G-CSF/IgG2b-Afllll/Hindlll) as described in Examples 1 & 2. The DNA sequence of resulting gene, SEQ ID NO: 50, encodes the protein of SEQ ID NO: 60.

EXAMPLE 10 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 Ncol/Hindlll restriction fragment from pMON30238 into plasmid pMON30309 digested with Afllll/Hindlll (which contains G-CSF/IgG2b-

Afllll/Hindlll) 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 NO: 40. 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 Ncol/Hindlll 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 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 NO: 20, 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 15

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 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 Afllll and Hindlll. The following three DNA fragments were ligated together to form plasmid pMON46406: 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 Stul-Xmal fragment encoding the terminal 6 amino acids of EPO and a portion of the GlySer polypeptide linker sequence

3. The 3,052 bp Ncol-Xmal 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 Afllll and Hindlll and the vector portion was purified. This was ligated with the 423 bp Ncol-Hindlll 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 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 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 scope of the invention. It is intended that all such other examples be included within the scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: G. D. Searle Corporate Patent Department

(ii) TITLE OF THE INVENTION: Flt3 Ligand Chimeric

Protein

(iii) NUMBER OF SEQUENCES: 65

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: G. D. Searle Corporate Patent Department

(B) STREET: P.O. Box 55110

(C) CITY: Chicago

(D) STATE: IL

(E) COUNTRY: USA

(F) ZIP: 60680

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER:

(B) FILING DATE: 10-APR-1998

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 08/837,026

(B) FILING DATE: ll-APR-1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Bennett, Dennis A

(B) REGISTRATION NUMBER: 34,547

(C) REFERENCE/DOCKET NUMBER: C-3018/1 /PCT

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 314-737-6986

(B) TELEFAX: 314-737-6972

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide ( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 1 :

Gly Gly Gly Ser 1

(2) INFORMATION FOR SEQ ID NO : 2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 5 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 :

Gly Gly Gly Gly Ser

1 5

( 2 ) INFORMATION FOR SEQ ID NO : 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 :

Gly Gly Gly Gly Gly Ser 1 5

(2) INFORMATION FOR SEQ ID NO : 4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 2 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 :

Gly Ser 1

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3 -imino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Ala Gly Ser 1

(2) INFORMATION FOR SEQ ID NO : 6 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 36 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) 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 15

Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser

20 25 30

Gly Gly Gly Ser 35

( 2 ) INFORMATION FOR SEQ ID NO : 7 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 7 : lie Ser Glu Pro Ser Gly Pro lie Ser Thr lie Asn Pro Ser Pro Pro

1 5 10 15

Ser Lys Glu Ser His Lys Ser Pro 20

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: lie Glu Gly Arg lie Ser Glu Pro Ser Gly Pro lie Ser Thr lie Asn 1 5 10 15 Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro 20 25

(2) INFORMATION FOR SEQ ID NO : 9 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 349 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser Asp Phe

1 5 10 15

Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro

20 25 30

Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu

35 40 45

Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val

50 55 60

Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu lie 65 70 75 80

His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg

85 90 95

Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin

100 105 110

Leu Val Ala Leu Lys Pro Trp lie 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 lie Ser Thr lie 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 lie 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 lie 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 lie 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 lie 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

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1047 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

GCCACCCAGG ACTGCTCCTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCATGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC 480

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACACC ATTGGGCCCT 540

GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG AAAGATCCAG 600

GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG 660

GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC 720

AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC 780

CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA 840

CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA 900

ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTC TGCTTTCCAG 960

CGCCGGGCAG GAGGGGTCCT GGTTGCTAGC CATCTGCAGA GCTTCCTGGA GGTGTCGTAC 1020

CGCGTTCTAC GCCACCTTGC GCAGCCC 1047

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 349 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu

1 5 10 15

Lys Ser Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu

20 25 30

Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu

35 40 45

Val Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu Ser Ser

50 55 60

Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His 65 70 75 80

Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie

85 90 95

Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala

100 105 110

Asp Phe Ala Thr Thr lie 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 lie Ser

180 185 190

Thr lie 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 lie Ser Ser Asp

210 215 220

Phe Ala Val Lys lie 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 lie His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu

290 295 300

Arg Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu 305 310 315 320

Gin Leu Val Ala Leu Lys Pro Trp lie 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

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1047 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG 60

CAAGTGAGAA 'AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC 120

AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT 180

CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT 240

AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG 300

GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG 360

CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC 420

TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC 480

TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTACGT AGAGGGCGGT 540

GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT 600

AAAGAATCTC ATAAATCTCC AAACATGGCT ACCCAGGACT GCTCCTTCCA ACACAGCCCC 660

ATCTCCTCCG ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCT TCAAGATTAC 720

CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGGCCT CTGGCGGCTG 780

GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGGTCCAA GATGCAAGGC 840

TTGCTGGAGC GCGTGAACAC GGAGATACAC TTTGTCACCA AATGTGCCTT TCAGCCCCCC 900

CCCAGCTGTC TTCGCTTCGT CCAGACCAAC ATCTCCCGCC TCCTGCAGGA GACCTCCGAG 960

CAGCTGGTGG CGCTGAAGCC CTGGATCACT CGCCAGAACT TCTCCCGGTG CCTGGAGCTG 1020

CAGTGTCAGC CCGACTCCTC AACCCTG 1047

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 798 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT 60

TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT 120

CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT 180

ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT 240 CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC 300

TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC 360

CCGGGTGGTG GTTCTGGCGG CGGCTCCAAC ATGGCCACCC AGGACTGCTC CTTCCAACAC 420

AGCCCCATCT CCTCCGACTT CGCTGTCAAA ATCCGTGAGC TGTCTGACTA CCTGCTTCAA 480

GATTACCCAG TCACCGTGGC CTCCAACCTG CAGGACGAGG AGCTCTGCGG GGCGCTCTGG 540

CGGCTGGTCC TGGCACAGCG CTGGATGGAG CGGCTCAAGA CTGTCGCTGG GTCCAAGATG 600

CAAGGCTTGC TGGAGCGCGT GAACACGGAG ATACACTTTG TCACCAAATG TGCCTTTCAG 660

CCCCCCCCCA GCTGTCTTCG CTTCGTCCAG ACCAACATCT CCCGCCTCCT GCAGGAGACC 720

TCCGAGCAGC TGGTGGCGCT GAAGCCCTGG ATCACTCGCC AGAACTTCTC CCGGTGCCTG 780

GAGCTGCAGT GTCAGCCC 798

(2) INFORMATION FOR SEQ ID NO: 14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 843 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT 60

TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT 120

CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT 180

ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT 240

CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC 300

TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC 360

CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT 420

CATAAATCTC CAAACATGGC CACCCAGGAC TGCTCCTTCC AACACAGCCC CATCTCCTCC 480

GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC 540

GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGCGC TCTGGCGGCT GGTCCTGGCA 600

CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG 660

CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT 720

CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG 780 GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG 340

CCC 343

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 813 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT 60

TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT 120

CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT 180

ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT 240

CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC 300

TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC 360

CCGGGTGGTG GTTCTGGCGG CGGCTCCAAC ATGGCCACCC AGGACTGCTC CTTCCAACAC 420

AGCCCCATCT CCTCCGACTT CGCTGTCAAA ATCCGTGAGC TGTCTGACTA CCTGCTTCAA 480

GATTACCCAG TCACCGTGGC CTCCAACCTG CAGGACGAGG AGCTCTGCGG GGCGCTCTGG 540

CGGCTGGTCC TGGCACAGCG CTGGATGGAG CGGCTCAAGA CTGTCGCTGG GTCCAAGATG 600

CAAGGCTTGC TGGAGCGCGT GAACACGGAG ATACACTTTG TCACCAAATG TGCCTTTCAG 660

CCCCCCCCCA GCTGTCTTCG CTTCGTCCAG ACCAACATCT CCCGCCTCCT GCAGGAGACC 720

TCCGAGCAGC TGGTGGCGCT GAAGCCCTGG ATCACTCGCC AGAACTTCTC CCGGTGCCTG 780

GAGCTGCAGT GTCAGCCCGA CTCCTCAACC CTG 813

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 858 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT 60 TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT 120

CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT 180

ATTGAGGCAA ' TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT 240

CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC 300

TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC 360

CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT 420

CATAAATCTC CAAACATGGC CACCCAGGAC TGCTCCTTCC AACACAGCCC CATCTCCTCC 480

GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC 540

GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGCGC TCTGGCGGCT GGTCCTGGCA 600

CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG 660

CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT 720

CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG 780

GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG 840

CCCGACTCCT CAACCCTG 858

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1047 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17:

GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG 60

CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC 120

AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT 180

CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT 240

AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG 300

GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG 360

CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC 420

TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC 480

TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTACGT AGAGGGCGGT 540 GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT 600

AAAGAATCTC ATAAATCTCC AAACATGGCC ACCCAGGACT GCTCCTTCCA ACACAGCCCC 660

ATCTCCTCCG 'ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCT TCAAGATTAC 720

CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGGCCT CTGGCGGCTG 780

GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGGTCCAA GATGCAAGGC 840

TTGCTGGAGC GCGTGAACAC GGAGATACAC TTTGTCACCA AATGTGCCTT TCAGCCCCCC 900

CCCAGCTGTC TTCGCTTCGT CCAGACCAAC ATCTCCCGCC TCCTGCAGGA GACCTCCGAG 960

CAGCTGGTGG CGCTGAAGCC CTGGATCACT CGCCAGAACT TCTCCCGGTG CCTGGAGCTG 1020

CAGTGTCAGC CCGACTCCTC AACCCTG 1047

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 942 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18:

GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC 480

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACCCA GGACTGCTCC 540

TTCCAACACA GCCCCATCTC CTCCGACTTC GCTGTCAAAA TCCGTGAGCT GTCTGACTAC 600

CTGCTTCAAG ATTACCCAGT CACCGTGGCC TCCAACCTGC AGGACGAGGA GCTCTGCGGG 660

GGCCTCTGGC GGCTGGTCCT GGCACAGCGC TGGATGGAGC GGCTCAAGAC TGTCGCTGGG 720

TCCAAGATGC AAGGCTTGCT GGAGCGCGTG AACACGGAGA TACACTTTGT CACCAAATGT 780

GCCTTTCAGC CCCCCCCCAG CTGTCTTCGC TTCGTCCAGA CCAACATCTC CCGCCTCCTG 840 CAGGAGACCT CCGAGCAGCT GGTGGCGCTG AAGCCCTGGA TCACTCGCCA GAACTTCTCC 900

CGGTGCCTGG AGCTGCAGTG TCAGCCCGAC TCCTCAACCC TG 942

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1047 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

GCCACCCAGG ACTGCTCCTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCATGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC 480

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACACC ATTAGGCCCT 540

GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTGCTTAG AGCAAGTGAG GAAGATCCAG 600

GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG 660

GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC 720

AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC 780

CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA 840

CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA 900

ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTC TGCTTTCCAG 960

CGCCGGGCAG GAGGGGTCCT GGTTGCTAGC CATCTGCAGA GCTTCCTGGA GGTGTCGTAC 1020

CGCGTTTTAC GCCACCTTGC GCAGCCC 1047

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1003 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) 'SEQUENCE DESCRIPTION: SEQ ID NO: 20:

GGCCACTCAG GACTGCTCTT TTCAACACAG CCCCATCTCC TCCGACTTCG CTGTCAAAAT 60

CCGTGAGCTG TCTGACTACC TGCTTCAAGA TTACCCAGTC ACCGTGGCCT CCAACCTGCA 120

GGACGAGGAG CTCTGCGGGG GCCTCTGGCG GCTGGTCCTG GCACAGCGCT GGATGGAGCG 180

GCTCAAGACT GTCGCTGGGT CCAAGATGCA AGGCTTGCTG GAGCGCGTGA ACACGGAGAT 240

ACACTTTGTC ACCAAATGTG CCTTTCAGCC CCCCCCCAGC TGTCTTCGCT TCGTCCAGAC 300

CAACATCTCC CGCCTCCTGC AGGAGACCTC CGAGCAGCTG GTGGCGCTGA AGCCCTGGAT 360

CACTCGCCAG AACTTCTCCC GGTGCCTGGA GCTGCAGTGT CAGCCCGACT CCTCAACCCT 420

GTACGTAGAG GGCGGTGGAG GCTCCCCGGG TGGTGGTTCT GGCGGCGGCT CCAACATGGC 480

TACACCATTG GGCCCTGCCA GCTCCCTGCC CCAGAGCTTC CTGCTCAAGT CTTTAGAGCA 540

AGTGAGAAAG ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA 600

GCTGTGCCAC CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC CCTGGGCTCC 660

CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC TGCTTGAGCC AACTCCATAG 720

CGGCCTTTTC CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCC CCGAGTTGGG 780

TCCCACCTTG GACACACTGC AGCTGGACGT CGCCGACTTT GCCACCACCA TCTGGCAGCA 840

GATGGAAGAA CTGGGAATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT 900

CGCCTCTGCT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT 960

CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCG 1003

(2) INFORMATION FOR SEQ ID NO: 21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 843 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTAACCCT 60

TTGCTGGACC CGAACAACCT CAATTCTGAA GACATGGATA TCCTGATGGA ACGAAACCTT 120

CGAACTCCAA ACCTGCTCGC ATTCGTAAGG GCTGTCAAGC ACTTAGAAAA TGCATCAGGT 180 ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT 240

CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC 300

TATCTGGTTA 'CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC 360

CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT 420

CATAAATCTC CAAACATGGC GACTCAGGAC TGTTCTTTCC AACACAGCCC CATCTCCTCC 480

GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC 540

GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGGCC TCTGGCGGCT GGTCCTGGCA 600

CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG 660

CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT 720

CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG 780

GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG 840

CCC 843

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 858 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:

GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTAACCCT 60

TTGCTGGACC CGAACAACCT CAATTCTGAA GACATGGATA TCCTGATGGA ACGAAACCTT 120

CGAACTCCAA ACCTGCTCGC ATTCGTAAGG GCTGTCAAGC ACTTAGAAAA TGCATCAGGT 180

ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT 240

CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC 300

TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC 360

CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT 420

CATAAATCTC CAAACATGGC AACCCAGGAC TGCTCTTTTC AACACAGCCC CATCTCCTCC 480

GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC 540

GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGGCC TCTGGCGGCT GGTCCTGGCA 600

CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG 660 CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT 720

CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG 780

GCGCTGAAGC 'CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG 840

CCCGACTCCT CAACCCTG 858

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 843 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:

GCCACTCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCTACGT AGAGGGCGGT 420

GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT 480

AAAGAATCTC ATAAATCTCC AAACATGGCT AACTGCTCTA TAATGATCGA TGAAATTATA 540

CATCACTTAA AGAGACCACC TAACCCTTTG CTGGACCCGA ACAACCTCAA TTCTGAAGAC 600

ATGGATATCC TGATGGAACG AAACCTTCGA ACTCCAAACC TGCTCGCATT CGTAAGGGCT 660

GTCAAGCACT TAGAAAATGC ATCAGGTATT GAGGCAATTC TTCGTAATCT CCAACCATGT 720

CTGCCCTCTG CCACGGCCGC ACCCTCTCGA CATCCAATCA TCATCAAGGC AGGTGACTGG 780

CAAGAATTCC GGGAAAAACT GACGTTCTAT CTGGTTACCC TTGAGCAAGC GCAGGAACAA 840

CAG 843

(2) INFORMATION FOR SEQ ID NO: 24:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 858 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24:

GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC 480

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTAACTG CTCTATAATG 540

ATCGATGAAA TTATACATCA CTTAAAGAGA CCACCTAACC CTTTGCTGGA CCCGAACAAC 600

CTCAATTCTG AAGACATGGA TATCCTGATG GAACGAAACC TTCGAACTCC AAACCTGCTC 660

GCATTCGTAA GGGCTGTCAA GCACTTAGAA AATGCATCAG GTATTGAGGC AATTCTTCGT 720

AATCTCCAAC CATGTCTGCC CTCTGCCACG GCCGCACCCT CTCGACATCC AATCATCATC 780

AAGGCAGGTG ACTGGCAAGA ATTCCGGGAA AAACTGACGT TCTATCTGGT TACCCTTGAG 840

CAAGCGCAGG AACAACAG 858

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 939 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 25:

GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360 ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GGTGGTTCTG GCGGCGGCTC CAACATGGCG 480

TCTCCGGCGC 'CGCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT 540

GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG 600

CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA 660

CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA 720

CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC 780

CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT 840

CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT 900

TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGG 939

(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 996 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 26:

GCGTCCCCAG CTCCACCTGC TTGTGACCTC CGAGTCCTCA GTAAACTGCT TCGTGACTCC 60

CATGTCCTTC ACAGCAGACT GAGCCAGTGC CCAGAGGTTC ACCCTTTGCC TACACCTGTC 120

CTGCTGCCTG CTGTGGACTT TAGCTTGGGA GAATGGAAAA CCCAGATGGA GGAGACCAAG 180

GCACAGGACA TTCTGGGAGC AGTGACCCTT CTGCTGGAGG GAGTGATGGC AGCACGGGGA 240

CAACTGGGAC CCACTTGCCT CTCATCCCTC CTGGGGCAGC TTTCTGGACA GGTCCGTCTC 300

CTCCTTGGGG CCCTGCAGAG CCTCCTTGGA ACCCAGCTTC CTCCACAGGG CAGGACCACA 360

GCTCACAAGG ATCCCAATGC CATCTTCCTG AGCTTCCAAC ACCTGCTCCG AGGAAAGGTG 420

CGTTTCCTGA TGCTTGTAGG AGGGTCCACC CTCTGCGTCA GGGAATTCCA TGCATACGTA 480

GAGGGCGGTG GAGGCTCCCC GGGTGAACCG TCTGGTCCAA TCTCTACTAT CAACCCGTCT 540

CCTCCGTCTA AAGAATCTCA TAAATCTCCA AACATGGCTA CCCAGGACTG CTCCTTCCAA 600

CACAGCCCCA TCTCCTCCGA CTTCGCTGTC AAAATCCGTG AGCTGTCTGA CTACCTGCTT 660

CAAGATTACC CAGTCACCGT GGCCTCCAAC CTGCAGGACG AGGAGCTCTG CGGGGGCCTC 720

TGGCGGCTGG TCCTGGCACA GCGCTGGATG GAGCGGCTCA AGACTGTCGC TGGGTCCAAG ATGCAAGGCT TGCTGGAGCG CGTGAACACG GAGATACACT TTGTCACCAA ATGTGCCTTT 840

CAGCCCCCCC CCAGCTGTCT TCGCTTCGTC CAGACCAACA TCTCCCGCCT CCTGCAGGAG 900

ACCTCCGAGC ' AGCTGGTGGC GCTGAAGCCC TGGATCACTC GCCAGAACTT CTCCCGGTGC 960

CTGGAGCTGC AGTGTCAGCC CGACTCCTCA ACCCTG 996

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1020 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:

GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC 480

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCCCCACC ACGCCTCATC 540

TGTGACAGCC GAGTCCTGGA GAGGTACCTC TTGGAGGCCA AGGAGGCCGA GAATATCACG 600

ACGGGCTGTG CTGAACACTG CAGCTTGAAT GAGAATATCA CTGTCCCAGA CACCAAAGTT 660

AATTTCTATG CCTGGAAGAG GATGGAGGTC GGGCAGCAGG CCGTAGAAGT CTGGCAGGGC 720

CTGGCCCTGC TGTCGGAAGC TGTCCTGCGG GGCCAGGCCC TGTTGGTCAA CTCTTCCCAG 780

CCGTGGGAGC CCCTGCAGCT GCATGTGGAT AAAGCCGTCA GTGGCCTTCG CAGCCTCACC 840

ACTCTGCTTC GGGCTCTGCG AGCCCAGAAG GAAGCCATCT CCCCTCCAGA TGCGGCCTCA 900

GCTGCTCCAC TCCGAACAAT CACTGCTGAC ACTTTCCGCA AACTCTTCCG AGTCTACTCC 960

AATTTCCTCC GGGGAAAGCT GAAGCTGTAC ACAGGGGAGG CCTGCAGGAC AGGGGACAGA 1020

(2) INFORMATION FOR SEQ ID NO: 28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 975 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28:

GCCCCACCAC GCCTCATCTG TGACAGCCGA GTCCTGGAGA GGTACCTCTT GGAGGCCAAG 60

GAGGCCGAGA ATATCACGAC GGGCTGTGCT GAACACTGCA GCTTGAATGA GAATATCACT 120

GTCCCAGACA CCAAAGTTAA TTTCTATGCC TGGAAGAGGA TGGAGGTCGG GCAGCAGGCC 180

GTAGAAGTCT GGCAGGGCCT GGCCCTGCTG TCGGAAGCTG TCCTGCGGGG CCAGGCCCTG 240

TTGGTCAACT CTTCCCAGCC GTGGGAGCCC CTGCAGCTGC ATGTGGATAA AGCCGTCAGT 300

GGCCTTCGCA GCCTCACCAC TCTGCTTCGG GCTCTGCGAG CCCAGAAGGA AGCCATCTCC 360

CCTCCAGATG CGGCCTCAGC TGCTCCACTC CGAACAATCA CTGCTGACAC TTTCCGCAAA 420

CTCTTCCGAG TCTACTCCAA TTTCCTCCGG GGAAAGCTGA AGCTGTACAC AGGGGAGGCC 480

TGCAGGACAG GGGACAGATA CGTAGAGGGC GGTGGAGGCT CCCCGGGTGG TGGTTCTGGC 540

GGCGGCTCCA ACATGGCCAC TCAGGACTGC TCTTTTCAAC ACAGCCCCAT CTCCTCCGAC 600

TTCGCTGTCA AAATCCGTGA GCTGTCTGAC TACCTGCTTC AAGATTACCC AGTCACCGTG 660

GCCTCCAACC TGCAGGACGA GGAGCTCTGC GGGGGCCTCT GGCGGCTGGT CCTGGCACAG 720

CGCTGGATGG AGCGGCTCAA GACTGTCGCT GGGTCCAAGA TGCAAGGCTT GCTGGAGCGC 780

GTGAACACGG AGATACACTT TGTCACCAAA TGTGCCTTTC AGCCCCCCCC CAGCTGTCTT 840

CGCTTCGTCC AGACCAACAT CTCCCGCCTC CTGCAGGAGA CCTCCGAGCA GCTGGTGGCG 900

CTGAAGCCCT GGATCACTCG CCAGAACTTC TCCCGGTGCC TGGAGCTGCA GTGTCAGCCC 960

GACTCCTCAA CCCTG 975

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 28 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 29:

GTAGTCCATG GCCACCCAGG ACTGCTCC 28

(2) INFORMATION FOR SEQ ID NO: 30: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 30 :

GCATTACGTA GGGCTGACAC TGCAGCTCCA G 1

(2) INFORMATION FOR SEQ ID NO: 31:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 31 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 31:

GCATTACGTA CAGGGTTGAG GAGTCGGGCT G 1

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 44 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32:

CCTGCAGGAC AGGGGACAGA TACGTAGAGG GCGGTGGAGG CTCC 4

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 48 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:

CCGGGGAGCC TCCACCGCCC TCTACGTATC TGTCCCCTGT CCTGCAGG 8

(2) INFORMATION FOR SEQ ID NO: 34: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 266 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:

Ala Asn Cys Ser lie Met lie Asp Glu lie lie His His Leu Lys Arg

1 5 10 15

Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val

20 25 30

Ser lie Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe

35 40 45

Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly lie Glu Ala lie

50 55 60

Leu Arg Asn Leu Gin Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser 65 70 75 80

Arg His Pro lie lie lie Lys Ala Gly Asp Trp Gin Glu Phe Arg Glu

85 90 95

Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gin Ala Gin Glu Gin Gin

100 105 110

Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly

115 120 125

Ser Asn Met Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser

130 135 140

Ser Asp Phe Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin 145 150 155 160

Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys

165 170 175

Gly Ala Leu Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu

180 185 190

Lys Thr Val Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn

195 200 205

Thr Glu lie His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser

210 215 220

Cys Leu Arg Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr 225 230 235 240

Ser Glu Gin Leu Val Ala Leu Lys Pro Trp lie Thr Arg Gin Asn Phe

245 250 255

Ser Arg Cys Leu Glu Leu Gin Cys Gin Pro 260 265

(2) INFORMATION FOR SEQ ID NO: 35:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 281 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 35: Ala Asn Cys Ser lie Met lie Asp Glu lie lie His His Leu Lys Arg 1 5 10 15

Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val

20 25 30

Ser lie Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe

35' 40 45

Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly lie Glu Ala lie

50 55 60

Leu Arg Asn Leu Gin Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser 65 70 75 80

Arg His Pro lie lie lie Lys Ala Gly Asp Trp Gin Glu Phe Arg Glu

85 90 95

Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gin Ala Gin Glu Gin Gin

100 105 110

Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro lie

115 120 125

Ser Thr lie Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro

130 135 140

Asn Met Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser 145 150 155 160

Asp Phe Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp

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 lie 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 lie Ser Arg Leu Leu Gin Glu Thr Ser

245 250 255

Glu Gin Leu Val Ala Leu Lys Pro Trp lie Thr Arg Gin Asn Phe Ser

260 265 270

Arg Cys Leu Glu Leu Gin Cys Gin Pro 275 280

(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 271 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36:

Ala Asn Cys Ser lie Met lie Asp Glu lie lie His His Leu Lys Arg

1 5 10 15

Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val

20 25 30

Ser lie Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe

35 40 45

Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly lie Glu Ala lie

50 55 60

Leu Arg Asn Leu Gin Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser 65 70 75 80 Arg His Pro lie lie lie Lys Ala Gly Asp Trp Gin Glu Phe Arg Glu

85 90 95

Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gin Ala Gin Glu Gin Gin

100 105 110

Tyr Val Glύ Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly

115 120 125

Ser Asn Met Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser

130 135 140

Ser Asp Phe Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin 145 150 155 160

Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys

165 170 175

Gly Ala Leu Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu

180 185 190

Lys Thr Val Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn

195 200 205

Thr Glu lie His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser

210 215 220

Cys Leu Arg Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr 225 230 235 240

Ser Glu Gin Leu Val Ala Leu Lys Pro Trp lie Thr Arg Gin Asn Phe

245 250 255

Ser Arg Cys Leu Glu Leu Gin Cys Gin Pro Asp Ser Ser Thr Leu 260 265 270

(2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 286 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37:

Ala Asn Cys Ser lie Met lie Asp Glu lie lie His His Leu Lys Arg

1 5 10 15

Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val

20 25 30

Ser lie Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe

35 40 45

Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly lie Glu Ala lie

50 55 60

Leu Arg Asn Leu Gin Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser 65 70 75 80

Arg His Pro lie lie lie Lys Ala Gly Asp Trp Gin Glu Phe Arg Glu

85 90 95

Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gin Ala Gin Glu Gin Gin

100 105 110

Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro lie

115 120 125

Ser Thr lie Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro

130 135 140

Asn Met Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser 145 150 155 160

Asp Phe Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp 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 lie 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 lie Ser Arg Leu Leu Gin Glu Thr Ser

245 250 255

Glu Gin Leu Val Ala Leu Lys Pro Trp lie 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

(2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 349 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) 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 15

Lys Ser Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu

20 25 30

Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu

35 40 45

Val Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu Ser Ser

50 55 60

Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His 65 70 75 80

Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie

85 90 95

Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala

100 105 110

Asp Phe Ala Thr Thr lie 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 lie Ser

180 185 190

Thr lie 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 lie Ser Ser Asp

210 215 220

Phe Ala Val Lys lie 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 lie His Phe Val Thr Lys Cys Ala Phe Gin ^'Pro Pro Pro Ser Cys Leu

290 295 300

Arg Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu 305 310 315 320

Gin Leu Val Ala Leu Lys Pro Trp lie 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

(2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 314 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO-.39:

Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser Asp Phe

1 5 10 15

Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro

20 25 30

Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu

35 40 45

Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val

50 55 60

Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu lie 65 70 75 80

His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg

85 90 95

Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin

100 105 110

Leu Val Ala Leu Lys Pro Trp lie 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 lie Ser Thr lie 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 lie Ser Ser Asp Phe Ala Val

180 185 190

Lys lie 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 lie His Phe 245 250 255

Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg Phe Val

260 265 270

Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin Leu Val

275 280 285

Ala Leu Lys Pro Trp lie Thr Arg Gin Asn Phe Ser Arg Cys Leu Glu

290 295 300

Leu Gin Cys Gin Pro Asp Ser Ser Thr Leu 305 310

(2) INFORMATION FOR SEQ ID NO: 40:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 349 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser Asp Phe

1 5 10 15

Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro

20 25 30

Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu

35 40 45

Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val

50 55 60

Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu lie 65 70 75 80

His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg

85 90 95

Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin

100 105 110

Leu Val Ala Leu Lys Pro Trp lie 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 lie Ser Thr lie 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 Cys

180 185 190

Leu Glu Gin Val Arg Lys lie 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 lie 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 lie 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 lie 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

(2) INFORMATION FOR SEQ ID NO: 41:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 334 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41:

Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser Asp Phe

1 5 10 15

Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro

20 25 30

Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu

35 40 45

Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val

50 55 60

Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu lie 65 70 75 80

His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg

85 90 95

Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin

100 105 110

Leu Val Ala Leu Lys Pro Trp lie 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 Gly Gly Ser Gly Gly Gly Ser Asn Met Ala 145 150 155 160

Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu Lys

165 170 175

Ser Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu Gin

180 185 190

Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val

195 200 205

Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu Ser Ser Cys

210 215 220

Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His Ser 225 230 235 240

Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie Ser

245 250 255

Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala Asp

260 265 270

Phe Ala Thr Thr lie Trp Gin Gin Met Glu Glu Leu Gly Met Ala Pro

275 280 285

Ala Leu Gin Pro Thr Gin Gly Ala Met Pro Ala Phe Ala Ser Ala Phe 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

(2) INFORMATION FOR SEQ ID NO: 42:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 281 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 42:

Ala Asn Cys Ser lie Met lie Asp Glu lie lie His His Leu Lys Arg

1 5 10 15

Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met

20 25 30

Asp lie Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe

35 40 45

Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly lie Glu Ala lie

50 55 60

Leu Arg Asn Leu Gin Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser 65 70 75 80

Arg His Pro lie lie lie Lys Ala Gly Asp Trp Gin Glu Phe Arg Glu

85 90 95

Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gin Ala Gin Glu Gin Gin

100 105 110

Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro lie

115 120 125

Ser Thr lie Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro

130 135 140

Asn Met Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser 145 150 155 160

Asp Phe Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp

165 170 175

Tyr Pro Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly

180 185 190

Gly 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 lie 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 lie Ser Arg Leu Leu Gin Glu Thr Ser

245 250 255

Glu Gin Leu Val Ala Leu Lys Pro Trp lie Thr Arg Gin Asn Phe Ser

260 265 270

Arg Cys Leu Glu Leu Gin Cys Gin Pro 275 280

(2) INFORMATION FOR SEQ ID NO: 43:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 286 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:

Ala Asn Cys Ser lie Met lie Asp Glu lie lie His His Leu Lys Arg

1 5 10 15

Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met

20 25 30

Asp lie Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe

35 40 45

Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly lie Glu Ala lie

50 55 60

Leu Arg Asn Leu Gin Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser 65 70 75 80

Arg His Pro lie lie lie Lys Ala Gly Asp Trp Gin Glu Phe Arg Glu

85 90 95

Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gin Ala Gin Glu Gin Gin

100 105 110

Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro lie

115 120 125

Ser Thr lie Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro

130 135 140

Asn Met Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser 145 150 155 160

Asp Phe Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp

165 170 175

Tyr Pro Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly

180 185 190

Gly 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 lie 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 lie Ser Arg Leu Leu Gin Glu Thr Ser

245 250 255

Glu Gin Leu Val Ala Leu Lys Pro Trp lie 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

(2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 281 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 44: Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser Asp Phe

1 5 10 15

Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro

20 25 30

Val Thr Vaϊ Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu

35 40 45

Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val

50 55 60

Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu lie 65 70 75 80

His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg

85 90 95

Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin

100 105 110

Leu Val Ala Leu Lys Pro Trp lie Thr Arg Gin Asn Phe Ser Arg Cys

115 120 125

Leu Glu Leu Gin Cys Gin Pro Tyr Val Glu Gly Gly Gly Gly Ser Pro

130 135 140

Gly Glu Pro Ser Gly Pro lie Ser Thr lie Asn Pro Ser Pro Pro Ser 145 150 155 160

Lys Glu Ser His Lys Ser Pro Asn Met Ala Asn Cys Ser lie Met lie

165 170 175

Asp Glu lie lie His His Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp

180 185 190

Pro Asn Asn Leu Asn Ser Glu Asp Met Asp lie Leu Met Glu Arg Asn

195 200 205

Leu Arg Thr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu

210 215 220

Glu Asn Ala Ser Gly lie Glu Ala lie Leu Arg Asn Leu Gin Pro Cys 225 230 235 240

Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro lie lie lie Lys

245 250 255

Ala Gly Asp Trp Gin Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val

260 265 270

Thr Leu Glu Gin Ala Gin Glu Gin Gin 275 280

(2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 286 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 45:

Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser Asp Phe

1 5 10 15

Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro

20 25 30

Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu

35 40 45

Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val

50 55 60

Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu lie 65 70 75 80

His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg

85 90 95

Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin

' 100 105 110

Leu Val Ala Leu Lys Pro Trp lie 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 lie Ser Thr lie Asn 145 150 155 160

Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Asn

165 170 175

Cys Ser lie Met lie Asp Glu lie lie His His Leu Lys Arg Pro Pro

180 185 190

Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met Asp lie

195 200 205

Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe Val Arg

210 215 220

Ala Val Lys His Leu Glu Asn Ala Ser Gly lie Glu Ala lie Leu Arg 225 230 235 240

Asn Leu Gin Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His

245 250 255

Pro lie lie lie Lys Ala Gly Asp Trp Gin Glu Phe Arg Glu Lys Leu

260 265 270

Thr Phe Tyr Leu Val Thr Leu Glu Gin Ala Gin Glu Gin Gin 275 280 285

(2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 313 -imino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46:

Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser Asp Phe

1 5 10 15

Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro

20 25 30

Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu

35 40 45

Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val

50 55 60

Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu lie 65 70 75 80

His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg

85 90 95

Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin

100 105 110

Leu Val Ala Leu Lys Pro Trp lie 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 Gly Gly Ser Gly Gly Gly Ser Asn Met Ala 145 150 155 160

Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu

165 170 175

Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu Val

180 185 190

His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu

195 200 205

Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp lie Leu

210 215 220

Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gin 225 230 235 240

Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly Gin

245 250 255

Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin Leu

260 265 270

Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala lie Phe

275 280 285

Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu

290 295 300

Val Gly Gly Ser Thr Leu Cys Val Arg 305 310

(2) INFORMATION FOR SEQ ID NO: 47:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 332 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 47:

Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu

1 5 10 15

Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gin Cys Pro Glu

20 25 30

Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser

35 40 45

Leu Gly Glu Trp Lys Thr Gin Met Glu Glu Thr Lys Ala Gin Asp lie

50 55 60

Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly 65 70 75 80

Gin Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gin Leu Ser Gly

85 90 95

Gin Val Arg Leu Leu Leu Gly Ala Leu Gin Ser Leu Leu Gly Thr Gin

100 105 110

Leu Pro Pro Gin Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala lie

115 120 125

Phe Leu Ser Phe Gin His Leu Leu Arg Gly Lys Val Arg Phe Leu Met

130 135 140

Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe His Ala Tyr Val 145 150 155 160

Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro lie Ser Thr

165 170 175 lie Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met 180 185 190

Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser Asp Phe

195 200 205

Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro

210 ' 215 220

Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu 225 230 235 240

Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val

245 250 255

Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu lie

260 265 270

His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg

275 280 285

Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin

290 295 300

Leu Val Ala Leu Lys Pro Trp lie Thr Arg Gin Asn Phe Ser Arg Cys 305 310 315 320

Leu Glu Leu Gin Cys Gin Pro Asp Ser Ser Thr Leu 325 330

(2) INFORMATION FOR SEQ ID NO: 48:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 340 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:

Ala Thr Gin Asp Cys Ser Phe Gin His Ser Pro lie Ser Ser Asp Phe

1 5 10 15

Ala Val Lys lie Arg Glu Leu Ser Asp Tyr Leu Leu Gin Asp Tyr Pro

20 25 30

Val Thr Val Ala Ser Asn Leu Gin Asp Glu Glu Leu Cys Gly Gly Leu

35 40 45

Trp Arg Leu Val Leu Ala Gin Arg Trp Met Glu Arg Leu Lys Thr Val

50 55 60

Ala Gly Ser Lys Met Gin Gly Leu Leu Glu Arg Val Asn Thr Glu lie 65 70 75 80

His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu Arg

85 90 95

Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu Gin

100 105 110

Leu Val Ala Leu Lys Pro Trp lie 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 lie Ser Thr lie Asn 145 150 155 160

Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Pro

165 170 175

Pro Arg Leu lie Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu

180 185 190

Ala Lys Glu Ala Glu Asn lie Thr Thr Gly Cys Ala Glu His Cys Ser 195 200 205 Leu Asn Glu Asn lie 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 lie Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu

290 295 300

Arg Thr lie 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

(2) INFORMATION FOR SEQ ID NO: 49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 325 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 49:

Ala Pro Pro Arg Leu lie Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu

1 5 10 15

Leu Glu Ala Lys Glu Ala Glu Asn lie Thr Thr Gly Cys Ala Glu His

20 25 30

Cys Ser Leu Asn Glu Asn lie Thr Val Pro Asp Thr Lys Val Asn Phe

35 40 45

Tyr Ala Trp Lys Arg Met Glu Val Gly Gin Gin Ala Val Glu Val Trp

50 55 60

Gin Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gin Ala Leu 65 70 75 80

Leu Val Asn Ser Ser Gin Pro Trp Glu Pro Leu Gin Leu His Val Asp

85 90 95

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 lie Ser Pro Pro Asp Ala Ala Ser Ala Ala

115 120 125

Pro Leu Arg Thr lie 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 lie Ser Ser Asp Phe Ala Val Lys lie Arg Glu Leu

195 200 205

Ser Asp Tyr Leu Leu Gin Asp Tyr Pro Val Thr Val Ala Ser Asn Leu 210 215 220

Gin Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gin 225 230 235 240

Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gin Gly

245 250 255

Leu Leu Glu Arg Val Asn Thr Glu lie His Phe Val Thr Lys Cys Ala

260 265 270

Phe Gin Pro Pro Pro Ser Cys Leu Arg Phe Val Gin Thr Asn lie Ser

275 280 285

Arg Leu Leu Gin Glu Thr Ser Glu Gin Leu Val Ala Leu Lys Pro Trp

290 295 300 lie Thr Arg Gin Asn Phe Ser Arg Cys Leu Glu Leu Gin Cys Gin Pro 305 310 315 320

Asp Ser Ser Thr Leu 325

(2) INFORMATION FOR SEQ ID NO: 50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1032 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50:

GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG 60

CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC 120

AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT 180

CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT 240

AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG 300

GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG 360

CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC 420

TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC 480

TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTACGT AGAGGGCGGT 540

GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT 600

AAAGAATCTC ATAAATCTCC AAACATGGCC ACCCAGGACT GCTCCTTCCA ACACAGCCCC 660

ATCTCCTCCG ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCT TCAAGATTAC 720

CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGGCCT CTGGCGGCTG 780

GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGGTCCAA GATGCAAGGC 840

TTGCTGGAGC GCGTGAACAC GGAGATACAC TTTGTCACCA AATGTGCCTT TCAGCCCCCC 900 CCCAGCTGTC TTCGCTTCGT CCAGACCAAC ATCTCCCGCC TCCTGCAGGA GACCTCCGAG 960

CAGCTGGTGG CGCTGAAGCC CTGGATCACT CGCCAGAACT TCTCCCGGTG CCTGGAGCTG 1020

CAGTGTCAGC 'CC 1032

(2) INFORMATION FOR SEQ ID NO: 51:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1005 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 51:

ATGGCCACTC AGGACTGCTC TTTTCAACAC AGCCCCATCT CCTCCGACTT CGCTGTCAAA 60

ATCCGTGAGC TGTCTGACTA CCTGCTTCAA GATTACCCAG TCACCGTGGC CTCCAACCTG 120

CAGGACGAGG AGCTCTGCGG GGGCCTCTGG CGGCTGGTCC TGGCACAGCG CTGGATGGAG 180

CGGCTCAAGA CTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGT GAACACGGAG 240

ATACACTTTG TCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCG CTTCGTCCAG 300

ACCAACATCT CCCGCCTCCT GCAGGAGACC TCCGAGCAGC TGGTGGCGCT GAAGCCCTGG 360

ATCACTCGCC AGAACTTCTC CCGGTGCCTG GAGCTGCAGT GTCAGCCCGA CTCCTCAACC 420

CTGTACGTAG AGGGCGGTGG AGGCTCCCCG GGTGGTGGTT CTGGCGGCGG CTCCAACATG 480

GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG 540

CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC 600

AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT 660

CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT 720

AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG 780

GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG 840

CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC 900

TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC 960

TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCG 1005

(2) INFORMATION FOR SEQ ID NO: 52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 420 base pairs

(B) TYPE: nucleic acid (C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) ' SEQUENCE DESCRIPTION: SEQ ID NO:52:

GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420

(2) INFORMATION FOR SEQ ID NO: 53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 405 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:

GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGC GCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCC 405

(2) INFORMATION FOR SEQ ID NO: 54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 420 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 54: GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC 'TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420

(2) INFORMATION FOR SEQ ID NO: 55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 420 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420

(2) INFORMATION FOR SEQ ID NO: 56:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 942 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 56:

GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180 CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC ' GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC 480

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACCCA GGACTGCTCC 540

TTCCAACACA GCCCCATCTC CTCCGACTTC GCTGTCAAAA TCCGTGAGCT GTCTGACTAC 600

CTGCTTCAAG ATTACCCAGT CACCGTGGCC TCCAACCTGC AGGACGAGGA GCTCTGCGGG 660

GGCCTCTGGC GGCTGGTCCT GGCACAGCGC TGGATGGAGC GGCTCAAGAC TGTCGCTGGG 720

TCCAAGATGC AAGGCTTGCT GGAGCGCGTG AACACGGAGA TACACTTTGT CACCAAATGT 780

GCCTTTCAGC CCCCCCCCAG CTGTCTTCGC TTCGTCCAGA CCAACATCTC CCGCCTCCTG 840

CAGGAGACCT CCGAGCAGCT GGTGGCGCTG AAGCCCTGGA TCACTCGCCA GAACTTCTCC 900

CGGTGCCTGG AGCTGCAGTG TCAGCCCGAC TCCTCAACCC TG 942

(2) INFORMATION FOR SEQ ID NO: 57:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1003 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 57:

GGCCACTCAG GACTGCTCTT TTCAACACAG CCCCATCTCC TCCGACTTCG CTGTCAAAAT 60

CCGTGAGCTG TCTGACTACC TGCTTCAAGA TTACCCAGTC ACCGTGGCCT CCAACCTGCA 120

GGACGAGGAG CTCTGCGGGG GCCTCTGGCG GCTGGTCCTG GCACAGCGCT GGATGGAGCG 180

GCTCAAGACT GTCGCTGGGT CCAAGATGCA AGGCTTGCTG GAGCGCGTGA ACACGGAGAT 240

ACACTTTGTC ACCAAATGTG CCTTTCAGCC CCCCCCCAGC TGTCTTCGCT TCGTCCAGAC 300

CAACATCTCC CGCCTCCTGC AGGAGACCTC CGAGCAGCTG GTGGCGCTGA AGCCCTGGAT 360

CACTCGCCAG AACTTCTCCC GGTGCCTGGA GCTGCAGTGT CAGCCCGACT CCTCAACCCT 420

GTACGTAGAG GGCGGTGGAG GCTCCCCGGG TGGTGGTTCT GGCGGCGGCT CCAACATGGC 480

TACACCATTG GGCCCTGCCA GCTCCCTGCC CCAGAGCTTC CTGCTCAAGT CTTTAGAGCA 540

AGTGAGAAAG ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA 600 GCTGTGCCAC CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC CCTGGGCTCC 660

CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC TGCTTGAGCC AACTCCATAG 720

CGGCCTTTTC' CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCC CCGAGTTGGG 780

TCCCACCTTG GACACACTGC AGCTGGACGT CGCCGACTTT GCCACCACCA TCTGGCAGCA 840

GATGGAAGAA CTGGGAATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT 900

CGCCTCTGCT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT 960

CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCG 1003

(2) INFORMATION FOR SEQ ID NO: 58:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 858 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 58:

GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC 60

CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG 120

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG 180

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA 240

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC 300

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC 360

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG 420

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC 480

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTAACTG CTCTATAATG 540

ATCGATGAAA TTATACATCA CTTAAAGAGA CCACCTAACC CTTTGCTGGA CCCGAACAAC 600

CTCAATTCTG AAGACATGGA TATCCTGATG GAACGAAACC TTCGAACTCC AAACCTGCTC 660

GCATTCGTAA GGGCTGTCAA GCACTTAGAA AATGCATCAG GTATTGAGGC AATTCTTCGT 720

AATCTCCAAC CATGTCTGCC CTCTGCCACG GCCGCACCCT CTCGACATCC AATCATCATC 780

AAGGCAGGTG ACTGGCAAGA ATTCCGGGAA AAACTGACGT TCTATCTGGT TACCCTTGAG 840

CAAGCGCAGG AACAACAG 858

(2) INFORMATION FOR SEQ ID NO: 59: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 402 base pairs

(B) TYPE: nucleic acid (C)' STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 59:

ATGGCTCCAA TGACTCAGAC TACTTCTCTT AAGACTTCTT GGGTTAACTG CTCTAACATG 60

ATCGATGAAA TTATAACACA CTTAAAGCAG CCACCTTTGC CTTTGCTGGA CTTCAACAAC 120

CTCAATGGGG AAGACCAAGA CATTCTGATG GAAAATAACC TTCGAAGGCC AAACCTGGAG 180

GCATTCAACA GGGCTGTCAA GAGTTTACAG AATGCATCAG CAATTGAGAG CATTCTTAAA 240

AATCTCCTGC CATGTCTGCC CCTGGCCACG GCCGCACCCA CGCGACATCC AATCCATATC 300

AAGGACGGTG ACTGGAATGA ATTCCGTCGT AAACTGACCT TCTATCTGAA AACCTTGGAG 360

AACGCGCAGG CTCAACAGAC CACTCTGTCG CTAGCGATCT TT 402

(2) INFORMATION FOR SEQ ID NO: 60:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 344 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 60:

Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gin Ser Phe Leu Leu

1 5 10 15

Lys Ser Leu Glu Gin Val Arg Lys lie Gin Gly Asp Gly Ala Ala Leu

20 25 30

Gin Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu

35 40 45

Val Leu Leu Gly His Ser Leu Gly lie Pro Trp Ala Pro Leu Ser Ser

50 55 60

Cys Pro Ser Gin Ala Leu Gin Leu Ala Gly Cys Leu Ser Gin Leu His 65 70 75 80

Ser Gly Leu Phe Leu Tyr Gin Gly Leu Leu Gin Ala Leu Glu Gly lie

85 90 95

Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gin Leu Asp Val Ala

100 105 110

Asp Phe Ala Thr Thr lie 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 lie Ser

180 185 190

Thr lie 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 lie Ser Ser Asp

210 215 220

Phe Ala Val Lys lie 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 lie His Phe Val Thr Lys Cys Ala Phe Gin Pro Pro Pro Ser Cys Leu

290 295 300

Arg Phe Val Gin Thr Asn lie Ser Arg Leu Leu Gin Glu Thr Ser Glu 305 310 315 320

Gin Leu Val Ala Leu Lys Pro Trp lie Thr Arg Gin Asn Phe Ser Arg

325 330 335

Cys Leu Glu Leu Gin Cys Gin Pro 340

(2) INFORMATION FOR SEQ ID NO: 61:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 133 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) 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 15

Asn Met lie Asp Glu lie lie Thr His Leu Lys Gin Pro Pro Leu Pro

20 25 30

Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp lie Leu Met

35 40 45

Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val

50 55 60

Lys Ser Leu Gin Asn Ala Ser Ala lie Glu Ser lie Leu Lys Asn Leu 65 70 75 80

Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro lie

85 90 95

His lie Lys Asp Gly Asp Trp Asn Gly lie 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 lie Phe 130

(2) INFORMATION FOR SEQ ID NO: 62: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 287 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single (D)' TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 62:

Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser

1 5 10 15

Asn Met lie Asp Glu lie lie Thr His Leu Lys Gin Pro Pro Leu Pro

20 25 30

Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp lie Leu Met

35 40 45

Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val

50 55 60

Lys Ser Leu Gin Asn Ala Ser Ala lie Glu Ser lie Leu Lys Asn Leu 65 70 75 80

Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro lie

85 90 95

His lie Lys Asp Gly Asp Trp Asn Gly lie 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 lie Phe Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly

130 135 140

Gly Ser Gly Gly Gly Ser Asn Met Ala Thr Gin Asp Cys Ser Phe Gin 145 150 155 160

His Ser Pro lie Ser Ser Asp Phe Ala Val Lys lie Arg Glu Leu Ser

165 170 175

Asp Tyr Leu Leu Gin Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gin

180 185 190

Asp Glu Glu Leu Cys Gly Ala Leu Trp Arg Leu Val Leu Ala Gin Arg

195 200 205

Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gin Gly Leu

210 215 220

Leu Glu Arg Val Asn Thr Glu lie His Phe Val Thr Lys Cys Ala Phe 225 230 235 240

Gin Pro Pro Pro Ser Cys Leu Arg Phe Val Gin Thr Asn lie Ser Arg

245 250 255

Leu Leu Gin Glu Thr Ser Glu Gin Leu Val Ala Leu Lys Pro Trp lie

260 265 270

Thr Arg Gin Asn Phe Ser Arg Cys Leu Glu Leu Gin Cys Gin Pro 275 280 285

(2) INFORMATION FOR SEQ ID NO: 63:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 302 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: None

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 63: Pro Met Thr Gin Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser

1 5 10 15

Asn Met lie Asp Glu lie lie Thr His Leu Lys Gin Pro Pro Leu Pro

' 20 25 30

Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gin Asp lie Leu Met

35 40 45

Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val

50 55 60

Lys Ser Leu Gin Asn Ala Ser Ala lie Glu Ser lie Leu Lys Asn Leu 65 70 75 80

Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro lie

85 90 95

His lie Lys Asp Gly Asp Trp Asn Gly lie 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 lie Phe Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu

130 135 140

Pro Ser Gly Pro lie Ser Thr lie 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 lie Ser Ser Asp Phe Ala Val Lys lie 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 lie His Phe Val Thr Lys Cys Ala Phe Gin

245 250 255

Pro Pro Pro Ser Cys Leu Arg Phe Val Gin Thr Asn lie Ser Arg Leu

260 265 270

Leu Gin Glu Thr Ser Glu Gin Leu Val Ala Leu Lys Pro Trp lie Thr

275 280 285

Arg Gin Asn Phe Ser Arg Cys Leu Glu Leu Gin Cys Gin Pro 290 295 300

(2) INFORMATION FOR SEQ ID NO: 64:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 407 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 64:

CCATGGCTAA CTGCTCTATA ATGATCGATG AAATTATACA TCACTTAAAG AGACCACCTG 0

CACCTTTGCT GGACCCGAAC AACCTCAATG ACGAAGACGT CTCTATCCTG ATGGATCGAA 20

ACCTTCGACT TCCAAACCTG GAGAGCTTCG TAAGGGCTGT CAAGAACTTA GAAAATGCAT 80 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

(2) INFORMATION FOR SEQ ID NO: 65:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 452 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 65:

CCATGGCTAA CTGCTCTATA ATGATCGATG AAATTATACA TCACTTAAAG AGACCACCTG 60

CACCTTTGCT GGACCCGAAC AACCTCAATG ACGAAGACGT CTCTATCCTG ATGGATCGAA 120

ACCTTCGACT TCCAAACCTG GAGAGCTTCG TAAGGGCTGT CAAGAACTTA GAAAATGCAT 180

CAGGTATTGA GGCAATTCTT CGTAATCTCC AACCATGTCT GCCCTCTGCC ACGGCCGCAC 240

CCTCTCGACA TCCAATCATC ATCAAGGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA 300

CGTTCTATCT GGTTACCCTT GAGCAAGCGC AGGAACAACA GTACGTAGAG GGCGGTGGAG 360

GCTCCCCGGG TGAACCGTCT GGTCCAATCT CTACTATCAA CCCGTCTCCT CCGTCTAAAG 420

AATCTCATAA ATCTCCAAAC ATGTAAGGTA CC 452

Claims

WHAT IS CLAIMED IS:

1. A chimeric protein comprising a flt3 agonist .

2. A chimeric protein comprising a polypeptide having a formula selected from the group consisting of:

R1-L-R2, R2-L-F-1, Rl~R2, R2-L-R1. Met-Ala-Rl-L- R2,

Met-Ala-R2 -L-R╬╣ , Met-Ala-R╬╣-R2 , Met-Ala-R2 ~Rl , Met-Ri-L-R2 , Met-R2 ~L-R╬╣ , Met-R╬╣~R2 , Met-R2-Rl , Ala-R╬╣-L-R2 , Ala-R2 -L-R╬╣ , Ala-R╬╣-R2 and Ala-R2 ~ Ri ;

wherein in Ri is a flt3 ligand;

R2 is a hematopoietic growth factor; and

L is a linker capable of linking R o R2 ΓÇó

3. The chimeric protein of claim 2 wherein said hematopoietic growth factor is selected from the group consisting of:

GM-CSF, G-CSF, G-CSF Ser¹⁷, c-mpl ligand, M-CSF, 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, IL-16, LIF, flt3 ligand, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor, SCSF, SDF-1 and SCF.

4. The chimeric protein of claim 3 wherein said hematopoietic growth factor is selected from the group consisting of G-CSF or G-CSF Ser ⁷.

5. The chimeric protein of claim 4 selected from the group consisting of;

the protein having the sequence of SEQ ID NO: 9;

the protein having the sequence of SEQ ID NO: 11;

the protein having the sequence of SEQ ID NO: 38;

the protein having the sequence of SEQ ID NO: 40; and

the protein having the sequence of SEQ ID NO: 41.

6. The chimeric protein of claim 3 wherein the hematopoietic growth factor is GM-CSF.

7. The chimeric protein of claim 3 wherein the hematopoietic growth factor is EPO.

8. The chimeric protein of claim 7 selected from the group consisting of;

the protein having the sequence of SEQ ID NO: 48; and

the protein having the sequence of SEQ ID NO: 49.

9. The chimeric protein of claim 3 wherein the hematopoietic growth factor is flt3 ligand.

10. The chimeric protein of claim 8 having the sequence of SEQ ID NO: 39.

11. The chimeric protein of claim 3 wherein the hematopoietic growth factor is c-mpl ligand.

12. The chimeric protein of claim 11 selected from the group consisting of;

the protein having the sequence of SEQ ID NO: 46; and

the protein having the sequence of SEQ ID NO: 47.

13. The chimeric protein of claim 3 wherein the hematopoietic growth factor is IL-3.

14. The chimeric protein of claim 13 selected from the group consisting of;

the protein having the sequence of SEQ ID NO: 62; and

the protein having the sequence of SEQ ID NO: 63.

15. A pharmaceutical composition comprising a chimera protein of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 and a pharmaceutically acceptable carrier.

16. A pharmaceutical composition comprising a chimera protein of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 and a hematopoietic growth factor and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16 wherein said hematopoietic growth factor is selected from the group consisting of;

GM-CSF, G-CSF, G-CSF Ser¹⁷, c-mpl ligand, M-CSF, 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, IL-16, LIF, B-cell growth factor, B-cell differentiation factor, eosinophil differentiation factor, SCSF, SDF-1 and SCF.

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

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

20. A nucleic acid molecule encoding the chimera protein of claim 3.

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

22. A nucleic acid molecule encoding the chimera protein of claim 5.

23. A nucleic acid molecule of claim 22 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 ;

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 NO : 20.

24. A nucleic acid molecule encoding the chimera protein of claim 6.

25. A nucleic acid molecule encoding the chimera protein of claim 7.

26. A nucleic acid molecule of claim 25 selected from the group consisting of:

a DNA sequencing having the sequence of SEQ ID NO: 27; and

a DNA sequencing having the sequence of SEQ ID NO : 28.

27. A nucleic acid molecule encoding the chimera protein of claim 9.

28. A nucleic acid molecule of claim 27 having a DNA sequencing having of SEQ ID NO: 27.

29. A nucleic acid molecule encoding the chimera protein of claim 11.

30. A nucleic acid molecule of claim 29 selected from the group consisting of:

a DNA sequencing having the sequence of SEQ ID NO: 25; and

a DNA sequencing having the sequence of SEQ ID

NO : 26.

31. A nucleic acid molecule encoding the chimera protein of claim 13.

32. 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 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 in a manner allowing expression of said chimera protein and recovering said chimera protein.

33. 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, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14.

34. A method of increasing hematopoietic cell production in a mammal in need thereof comprising administering a pharmaceutically effective amount of the composition of claim 15.

35. A method of increasing hematopoietic cell production in a mammal in need thereof comprising administering a pharmaceutically effective amount of the composition of claim 16.

36. A method of increasing hematopoietic cell production in a mammal in need thereof comprising administering a pharmaceutically effective amount of the composition of claim 17.

37. A method for ex vivo expansion of stem cells, comprising the steps of;

(a) culturing said stem cells with a selected growth medium comprising a chimeric protein of claims 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14; and

(b) harvesting said cultured stem cells.

3,8. 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 37.

39. A method of human gene therapy, comprising the steps of;

(a) removing stem cells from a patient or donor

(b) culturing said stem cells with a selected growth medium comprising a chimera protein of claim 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14;

(c) transducing DNA into said cultured cells; (d) harvesting said transduced cells; and

(e) transplanting said transduced cells into said patient.