CA2284127A1 - 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 LiQaad 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,eukin-3 (IL-3) have broader activities and stimulate the formation of both macrophage, neutrophilic and eosinophilic granulocyte colonies.
Certain factors such as flt3 ligand are able to predominately affect stem cells.
Tyrosine kinase receptors are growth factor receptors that regulate the proliferation and differentiation of a number of cell. Certain tyrosine kinase receptors function within the hematopoietic system. Flt3 (Roseate et al., Oncogene, 6:1641-1650, 1991) and flk-2 (Matthews et al., Cell, 65:1143-1152, 1991) are forms of a tyrosine kinase receptor that is related to c-fms and c-kit receptors. The flk-2 and flt3 receptors are similar in amino acid sequence and vary at two amino acid residues in the extracellular domain and diverge in a 31 amino acid segment located near the C-terminus.
Flt3 ligand is a hematopoietic growth factor which has the property of being able to regulate the growth and differentiation of hematopoietic progenitor and stem cells. Because of its ability to support the growth and proliferation of progenitor cells, flt3 receptor agonists have potential for therapeutic use in treating hematopoietic disorders such as aplastic anemia and myelodysplastic syndromes. Additionally, flt3 receptor agonists will be useful in restoring hematopoietic cells to normal amounts in those cases where the number of cells has been reduced due to diseases or to therapeutic treatments such as radiation and chemotherapy.
WO 94/28391 discloses the native flt3 ligand protein sequence and a cDNA sequence encoding the flt3 ligand, methods of expressing flt3 ligand in a host cell transfected with the cDNA and methods of treating patients with a hematopoietic disorder using flt3 7,igand.
US Patent No. 5,554,512 is directed to human S flt3 ligand as an isolated protein, DNA encoding the flt3 ligand, host cells transfected with cDNAs encoding flt3 ligand and methods for treating patients with flt3 ligand.
WO 94/26891 provides mammalian flt3 ligands, including an isolate that has an insertion of 29 amino acids, and fragments there of.
The human blood-forming (hematopoietic) system replaces a variety of white blood cells (including neutrophils, macrophages, and basophils/mast cells), red blood cells (erythrocytes) and clot-forming cells (megakaryocytes/platelets). The hematopoietic systems of the average male has been estimated to produce on the order of 4.5 x 1011 granulocytes and erythrocytes every year, which is equivalent to an annual replacement of total body weight (Dexter et al., BioEssays, 2;154-158, 1985).
U.S. Patent 4,999,291 discloses DNA and methods for making G-CSF the disclosure of which is incorporated herein by reference in it entirety.
U.S. Patent 4,810,643 relates to DNA and methods of making G-CSF and Cys to Ser substitution variants of G-CSF.
Kuga et al. (Biochem. + Biophys. Res. Comm.
159:103-111, 1988) made a series of G-CSF variants to partially define the structure-function relationship.
Kuga et al. found that internal and C-terminal deletions abolished activity, while N-terminal deletions of up to 11 amino acids and amino acid substitutions at positions 1, 2 and 3 were active.
Watanabe et al. (Anal. Biochem. 195:38-44, 1991) made a variant to study G-CSF receptor binding in which amino acids 1 and 3 were changed to Tyr for radioiodination of the protein. Watanabe et al. found this Tyrl, 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 N0. 4,703,008, replacement of the tyrosine residues at positions 15, 40 and 145 with phenylalanine residues, replacement of the cysteine residue at position 7 with an histidine, substitution of the proline at position 2 with an asparagine, deletion of residues 2-6, deletion of residues 163-166, and deletion of residues 27-55 does not result in an apparent increase in biological activity. The Cys~-to-Hiss 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 5 the O-linked glycosylation site at serine126 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 vitro 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 erythz:opoietin (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 vitro bioassay while the domain 2 mutants, at best, retain in vitro activity. These mutants also show no enhanced in vivo biological activity as compared to wild-type, human erythropoietin. These authors 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 vitro activity. The loss of activity is so severe that the authors conclude that the disulfide bridge between residues 29 and 33 is essential for erythropoietin function (F.K. Lin, Molecular and Cellular Aspects of Erythropoietin and Erythropoiesis, pp. 23-36, ed. I.N. Rich, Springer-Verlag, Berlin (1987)).
U.S. Patent No. 4,703,008 by Lin, F-K.
(hereinafter referred to as "the '008 patent") speculates about a wide variety of modifications of EPO, including addition, deletion, and substitution analogs of EPO. The '008 patent does not indicate that any of the suggested modifications would increase biological activity per se, although it is stated that deletion of glycosylation sites might increase the activity of EPO produced in yeast (See the '008 patent at column 37, lines 25-28). Also, the '008 patent speculates that EPO analogs which . have one or more tyrosine residues replaced with phenylalanine may exhibit an increased or decreased receptor binding affinity.

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 (Asn6g), EPO {Asn125, 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 l~,rmphoid cells .
EP 0 423 980 discloses novel stem cell factor (SCF) polypeptides including SCFl'laa, SCF1-ls', SCF1-lso SCF1-isi, SCFl-isz, SCFl'isa, SCFi-iss, SCFl-ia3, SCFl'las~ SCFl' iaa, SCFl'ies, SCFl-zzo, SCFl-zaa , 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 Sera -> ProB replacement.
Suggestions are made to replace Cys by Ser, thereby breaking the disulfide bridge, and to replace one or more amino acids at the glycosylation sites.
U.S. 4,810,643 discloses the DNA sequence encoding human G-CSF.
WO 91/02754 discloses a fusion protein comprised of GM-CSF and IL-3 which has increased biological activity compared to GM-CSF or IL-3 alone. Also disclosed are nonglycosylated IL-3 and GM-CSF analog proteins as components of the multi-functional hematopoietic receptor agonist.
WO 92/04455 discloses fusion proteins composed of IL-3 fused to a lymphokine selected from the group consisting of IL-3, IL-6, IL-7, IL-9, IL-11, EPO and G-CSF.
. WO 95/21197 and WO 95/21254 disclose fusion proteins capable of broad multi-functional hematopoietic properties.
GB 2,285,446 relates to the c-mpl ligand (thrombopoietin) and various forms of thrombopoietin which are shown to influence the replication, WO 98/46?50 PCT/US98/0?511 differentiation and maturation of megakaryocytes and megakaryocytes progenitors which may be used for the treatment of thrombocytopenia.
EP 675,201 A1 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 factox(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 multi-functional hematopoietic receptor agonists or chimeric proteins formed from covalently linked polypeptides, each of which may act through a different and specific cell receptor to initiate complementary biological activities. Hematopoiesis requires a complex series of cellular events in which stem cells generate continuously into large populations of maturing cells in all major lineages.
There are currently at least 20 known regulators with WO 98/46750 PCTlUS98/07511 hematopoietic proliferative activity. Most of these proli~erative 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. eio. Chem. 265:14008-15, 1990), IL-4 (Harada et al., PNAS USA 87:857, 1990), IL-5 (Takaki et al., E1~0 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., E1~0 J.
10:2839, 1991) and IL-2 (Cosman et al. , Mo1-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 a-chains become associated with at least one other receptor chain (~i-chain, y-chain). Many of these factors share a common receptor subunit. The a-chains for GM-CSF, IL-3 and IL-5 share the same (3-chain (Kitamura et al., Cell 66:1165, 1991), Takaki et al., EM80 J. 10:2833-8, 1991) and receptor complexes for IL-6, LIF and IL-11 share a common (3-chain (gp130) (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 y-chain (Kondo et al., Science 262:1874, 1993; Russell WO 98/46750 PCT/US98/0'1511 et al., Science 266: 1042-1045, 1993; Noguchi et al., Science 262:1877, 1993; Giri et al., ~O 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 TnThere R1 is a flt3 agonist and R2 is a hematopoietic growth factor. Preferably R2 is a hematopoietic growth factor with a different but complementary activity than R1. By complementary activity is meant activity which enhances or changes the response to another cell modulator. The R1 polypeptide is joined either directly or through a linker segment to the R2 polypeptide. The term "directly" defines multi-functional hematopoietic receptor agonists in which the polypeptides are joined without a peptide linker. Thus L1 represents a chemical bond or polypeptide segment to which both R1 and R2 are joined in frame, most commonly L1 is a linear peptide to which R1 and R2 are joined by amide bonds linking the carboxy terminus of R1 to the amino terminus of L1 and carboxy terminus of L1 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 R1 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 R1 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 R1 or R2 molecules or mutated or modified DNA sequences encoding these R1 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 (L1) 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 R1 and R2 such that R1 and R2 could interact simultaneously with their corresponding receptors on a single cell. Typically surface amino acids in flexible protein regions include Gly, Asn and Ser. Virtually any permutation of amino acid sequences containing Gly, Asn and Ser would be expected to satisfy the above criteria for a linker sequence. Other neutral amino acids, such as Thr and Ala, may also be used in the linker sequence.
Additional amino acids may also be included in the 4linkers due to the addition of unique restriction sites in the linker sequence to facilitate construction of the multi-functional hematopoietic receptor agonists.
Preferred L1 linkers of the present invention include sequences selected from the group of formulas:
(Gly3Ser)n (SEQ ID N0:1), (Gly4Ser)n (SEQ ID N0:2), (GlySSer)n (SEQ ID N0:3), (GlynSer)n (SEQ ID N0:4) or (AlaGlySer)n (SEQ ID N0:5), where n is an integer (collectively referred to herein as "GlySer"
linkers).
One example of a highly-flexible linker is the glycine and serine-rich spacer region present within the pIII protein of the filamentous bacteriophages, e.g. bacteriophages M13 or fd (Schaller et al., PNAS
USA 72: 737-741, 1975). This region provides a long, flexible spacer region between two domains of the pIII surface protein. The spacer region consists of the amino acid sequence:
3 5 GlyGlyGlySerGlyGlyGlySerGlyGlyGlySerGluGlyGlyGlySerGlu GlyGlyGlySerGluGlyGlyGlySerGluGlyGlyGlySerGlyGlyGlySer (sEQ ID N0:6).

'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 multi-functional hematopoietic receptor agonist to determine if they are properly folded and active in vitro. Examples of various endopeptidases include, but are not limited to, plasmin, enterokinase, kallikrein, urokinase, tissue plasminogen activator, clostripain, chymosin, collagenase, Russell's viper venom protease, postproline cleavage enzyme, V8 protease, Thrombin and factor Xa.
Peptide linker segments from the hinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgE
provide an angular relationship between the attached polypeptides. Especially useful are those hinge regions where the cysteines are replaced with serines. Preferred linkers of the present invention include sequences derived from murine IgG gamma 2b hinge region in which the cysteines have been changed to serines (Bell et al. US Patent 4,936,233). These linkers may also include an endopeptidase cleavage site. Examples of such linkers include the following sequences:
IleSerGluProSerGlyProIleSerThrIleAsnProSerProProSerLys GluSerHisLysSerPro ( SEQ ID NO : 7 ) , and 3 0 IleGluGlyArgIleSerGluProSerGlyProIleSerThrIleAsnProSer ProProSerLysGluSerHisLysSerPro (SEQ ID N0:8) (COllectlVely 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 mqlti-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 multi-functional hematopoietic receptor agonists of the invention only due to the degeneracy of the genetic code. ~rlso 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 multi-functional 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 multi-functional 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 encode Met 2-Ala 1- or Met 1 at the N-terminus of the protein. The N termini of proteins made in the cytoplasm of E. coli are affected by post-translational processing by methionine aminopeptidase (Ben Bassat et al., J. Bac. 169:751-757, 1987) and possibly by other peptidases so that upon expression the methionine is cleaved off the N-terminus. The multi-functional hematopoietic receptor agonists of the present invention may include multi-functional hematopoietic receptor agonist polypeptides having Met 1, Ala 1 or Met 2-Ala 1 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 COS7, HeLa, BHK, CHO, or mouse L
lines. The cells can be cultured, for example, in DMEM media (JRH Scientific). The polypeptide secreted into the media can be recovered by standard biochemical approaches following transient expression for 24 - 72 hours after transfection of the cells or wo 9s~~w~so rc°rnJS9s~o~si i 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 multi-functional 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 multi-functional 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 (SLEy, 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 autalogous bone marrow transplants, some of the patient's own marrow is harvested prior to myeloablative therapy, e.g. high dose chemotherapy, and is transplanted back into the patient afterwards. Autologous transplants eliminate the risk of Graft Versus Host Disease and graft rejection. However, autologous bone marrow transplants still present problems in terms of the limited number of stems cells in the marrow and possible contamination with tumor cells. The limited number of stem cells may be overcome by ex-vivo expansion of the stem cells. In addition, stem cells can be specifically isolated, based on the presence of specific surface antigens such as CD34+ in order to decrease tumor cell contamination of the marrow graft.
The following patents contain further details on separating stem cells, CD34+ cells, culturing the cells with hematopoietic factors, the use of the cells for the treatment of patients with hematopoietic disorders and the use of hematopoietic factors for cell expansion and gene therapy.
5,061,620 relates to compositions comprising human hematopoietic stem cells provided by separating the stem cells from dedicated cells.

WO 98/46750 PC"f/US98/07511 5,199,942 describes a method for autologous hematppoietic 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 wo 9s~a6~so pc'r~s9a~o~sm myeloblasts and promyelocytes for treating genetic or acquired neutropenia.
WO 94/08039 describes a method of enrichment for human hematopoietic stem cells by selection for cells which express c-kit protein.
WO 94/11493 describes a stem cell population that are CD34+ and small in size, which are isolated using a counterflow elutriation method.
w0 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 proteins) 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-1). Other surface antigens such as CD33, CD38, CD41, CD71, HLA-DR or c-kit can also be used to selectively isolate hematopoietic progenitors. The separated cells can be incubated in selected medium in a culture flask, sterile bag or in hollow fibers. Various colony stimulating factors may be utilized in order to selectively expand cells.
Representative factors that have been utilized for ex-vivo expansion of bone marrow include, c-kit ligand, IL-3, G-CSF, GM-CSF, IL-1, IL-6, IL-11, 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., Fxp. Hem. 21:1668-1672 [1993], Sato et al., Blood 82:3600-3609 [1993]), flt3 ligand (McKenna et al., Blood 86:3413 3420 [1995]) and/or combinations thereof (Brandt et al., Blood 83:1507 1514 [1994], Haylock et al., Blood 80:1405-1412 [1992], Koller et al., Biotechnology 11:358-363 [1993], (Lemoli et al., Exp. Hem. 21:1668-1672 [1993]), McKenna et al., Blood 86:3413-3420 [1995], Muench et al., Blood 81:3463-3473 [1993], Patchen et al., Biotherapy 7:13-26 [1994], Sato et al., Blood 82:3600-3609 [1993], Smith et al., Exp. Hem. 21:870-877 [1993], Steen et al., Stem Cells 12:214-224 [1994], Tsujino et al., Exp. Hem. 21:1379-1386 [1993]). Among the individual colony stimulating factors, hIL-3 has been shown to be one of the most potent in expanding peripheral blood CD34+ cells (Sato et al., Blood 82:3600-3609 [1993], Kobayashi et al., Blood 73:1836-1841 [1989]). However, no single factor has been shown to be as effective as the combination of multiple factors. The present invention provides methods for ex vivo expansion that utilise 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 S material into a host cell. A number of vectors, both viral and non-viral have been developed for transferring therapeutic genes into primary cells.
Viral based vectors include; 1) replication deficient recombinant retrovirus (Boris-Lawrie and Temin, Curr.
Opin. Genet. Dev. 3:102-109 [1993], Boris-Lawrie and Temin, Annal. New York Acad. Sci. 716:59-71 [1994], Miller, Current Top. Microbiol. Immunol. 158:1-24 [1992]) and replication-deficient recombinant adenovirus (Berkner, BioTechniques 6:616-629 [1988], Berkner, Current Top. Microbiol. Imniunol. 158:39-66 [1992], Brody and Crystal, Arsnal. 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 dipyrpne, 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.

' 35 -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 mufti-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 mufti-functional hematopoietic receptor agonists to a patient. The mufti-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 mufti-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 ug/kg of mufti-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 multi-functional 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 mufti-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
Serb , 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 roteins Biological activity of the multi-functional hematopoietic receptor agonist proteins of the present invention can be determined by DNA synthesis in factor-dependent cell lines or by counting the colony forming units in an in vitro bone marrow assay. The chimeric proteins may be assayed by a number of in vitro and in vivo models known to those skilled in the art. Examples of such assays include put are not limited to:
Methylcellulose Assay This assay reflects the ability of colony stimulating factors to stimulate normal bone marrow cells to produce different types of hematopoietic colonies in vitro (Bradley et al., Aust. Exp Biol. Sci. 44:287-300, 1966), Pluznik et al., J. Cell Comp. Physio 66:319-324, 1965).
Methods Approximately 30 mL of fresh, normal, healthy bone marrow aspirate are obtained from individuals following informed consent. Under sterile conditions samples are diluted 1:5 with a 1X PBS (#14040.059 Life Technologies, Gaithersburg, MD.) solution in a 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 1X 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 multi-functional 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 multi-functional 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 (Bro~neyer 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 lml of 0.9~
5 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 10 (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, 198?). The ability of AML 193 cells to proliferate in the presence of human IL-3 has also been documented. (Santoli, D., et al., J. Immunol. 139: 348, 1987). A cell line variant was used, AML 193 1.3, which was adapted for long term growth in IL-3 by washing out the growth factors and starving the cytokine dependent AML 193 cells for growth factors for 24 hours. The cells are then replated at 1x105 cells/well in a 24 well plate in media containing 100 U/mL IL-3. It took approximately 2 months for the cells to grow rapidly in IL-3. These cells are maintained as AML 193 1.3 WO 98/46750 PC"T/US98/07511 thereafter by supplementing tissue culture medium (see below) with human IL-3.
AML 193 1.3 cells are washed 6 times in cold Hanks balanced salt solution (HBSS, Gibco, Grand Island, NY) by centrifuging cell suspensions at 250 x g for 10 minutes followed by decantation of the supernatant. Pelleted cells are resuspended in HBSS
and the procedure is repeated until six wash cycles are completed. Cells washed six times by this procedure are resuspended in tissue culture medium at a density ranging from 2 x 105 to 5 x 105 viable cells/mL. This medium is prepared by supplementing Iscove's modified Dulbecco's Medium (IMDM, Hazelton, Lenexa, KS) with albumin, transferrin, lipids and 2-mercaptoethanol. Bovine albumin (Boehringer-Mannheim, Indianapolis, IN) is added at 500 ug/mL;
human transferrin (Boehringer-Mannheim, Indianapolis, IN) is added at 100 ug/mL; soybean lipid (Boehringer-Mannheim, Indianapolis, IN) is added at 50 ug/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 ul 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 50 ul (2.5 x 104 cells) into each well. Tissue culture plates are incubated at 37°C with 5~ C02 in humidified air for 3 days. On day 3, 0.5 uCi 3H-thymidine (2 Ci/mM, New England Nuclear, Boston, MA) is added in 50 ul of tissue culture medium. Cultures are incubated at 37°C with 5~ C02 in humidified air for 18-24 hours. Cellular DNA is harvested onto glass filter mats (Pharmacia LKB, Gaithersburg, MD) using a TOMTEC cell harvester (TOMTEC, Orange, CT) which utilized a water wash cycle followed by a 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.
Typically, concentration ranges from 0.05 pM - 105 pM
are quantitated in these assays. Activity is determined by measuring the dose of interleukin-3 or multi-functional chimeric hematopoietic receptor agonist protein which provides 50~ of maximal proliferation (ECSO = 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 ECSo value is also equivalent to 1 unit of bioactivity.
Every assay is performed with native interleukin-3 as a reference standard so that relative activity levels could be assigned.
Typically, the multi-functional chimeric hematopoietic receptor agonist proteins were tested in a concentration range of 2000 pM to 0.06 pM
titrated in serial 2 fold dilutions.
Activity for each sample was determined by the concentration which gave 50~ of the maximal response by fitting a four-parameter logistic model to the data. It was observed that the upper plateau (maximal response) for the sample and the standard with which it was compared did not differ. Therefore relative potency calculation for each sample was determined from EC50 estimations for the sample and the standard as indicated above. AML 193.1.3 cells proliferate in response to hIL-3, hGM-CSF and hG-CSF.
TF1 c-mpl ligand dependent proliferation assay The c-mpl ligand proliferative activity can be assayed using a subclone of the pluripotential human cell line TF1 (Kitamura et al., J. Cell Physiol 140:323-334. [1989]). TF1 cells are maintained in h-IL3 (100 U/mL?. To establish a sub-clone responsive to c-mpl ligand, cells are maintained in passage media containing 10$ supernatant from BHK cells transfected with the gene expressing the 1-153 form of c-mpl ligand (pMON26448). Most of the cells die, but a subset of cells survive. After dilution cloning, a c-mpl ligand responsive clone is selected, and these cells are split into passage media to a density of 0.3 x 106 cells/mL the day prior to assay ' 44 -set-up. Passage media for these cells is the following: RPMI 1640 (Gibco), 10~ FBS (Harlan, Lot #91206), 10$ c-mpl ligand supernatant from transfected BHK cells, 1 mM sodium pyruvate (Gibco), 2 mM glutamine (Gibco), and 100 ug/mL 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 106 cells/mL
in a 96-well low evaporation plate (Costar) to a final volume of 50 ul. Transient supernatants (conditioned media) from transfected clones are added at a volume of 50 ul as duplicate samples at a final concentration of 50~ and diluted three-fold to a final dilution of 1.8~. Triplicate samples of a dose curve of IL-3 variant pMON13288 starting at 1 ng/mL
and diluted using three-fold dilutions to 0.0014ng/mL
is included as a positive control. Plates are incubated at 5~ C02 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~ C02 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 (20-100ng/mL) in the growth medium. Eighteen hours prior to assay set-up, MUTZ-2 cells are washed in IMDM
medium (Gibco) three times and are resuspended in IMDM medium alone at a concentration of 0.5-0.7 x 10E6 cells/mL and incubated at 37°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. 50u1 of assay media is loaded into all wells except row A. 75u1 of the flt3 receptor agonists or standards are added to row A and 25u1 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 50u1 assay media. 50u1 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 luCi/well of tritiated thymidine in a volume of 20u1 for four hours. Plates are then harvested and counted.
Other in vitro cell based proliferation assays ' 46 -Other in vitro cell based assays, known to those skilled in the art, may also be useful to determine the activity of the multi-functional chimeric hematopoietic receptor agonists depending on the factors that comprise the molecule in a similar manner as described in the AML 193.1.3 cell proliferation assay. The following are examples of other useful assays.
TF1 proliferation assay: TF1 is a pluripotential human cell line (Kitamura et al., J. Cell Physiol 140:323-334. [1989]) that responds to hIL-3.
32D proliferation assay: 32D is a murine IL-3 dependent cell line which does not respond to human IL-3 but does respond to human G-CSF which is not species restricted.
Baf/3 proliferation assay: Baf/3 is a murine IL-3 dependent cell line which does not respond to human IL-3, human flt3 ligand or human c-mpl ligand but does respond to human G-CSF which is not species restricted.
T1165 proliferation assay: T1165 cells are a IL-6 dependent murine cell line (Nordan et al., 1986) which respond to IL-6 and IL-11.
Human Plasma Clot meg-CSF Assay: Used to assay megakaryocyte colony formation activity (Mazur et al., 1981).
Transfected cell lines:
Cell lines such as the murine Baf/3 cell line can be transfected with a hematopoietic growth factor 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 6418 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 I5 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 48 ' instructions (CellPro, Inc., Bothell WA). After enrichment, the purity of CD34+ cells was 70~ on average as determined by using flow cytometric analysis using anti-CD34 monoclonal antibody conjugated to fluorescein and anti-CD38 conjugated to phycoerythrin (Becton Dickinson, San Jose CA).
Cells were resuspended at 40,000 cells/mL in X-Vivo 10 media (Bio-Whittaker, Walkersville, MD) and 1 mL was plated in 12-well tissue culture plates (Costar). The growth factor rhIL-3 was added at 100 ng/mL (pMON5873) was added to some wells. hIL3 variants were used at 10 ng/mL to 100 ng/mL.
Conditioned media from BHK cells transfected with plasmid encoding c-mpl ligand or multi-functional chimeric hematopoietic receptor agonists were tested by addition of 100 ul 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 dun PGE1, 11 mM glucose, 3~ w/v BSA, in PBS, pH 7.4,) (Tourer et al., Blood 70: 1735-1742, 1987) resuspended in 500 ul of MK buffer containing anti-CD4la 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 WO 98/46750 PGT/~JS98/07511 (Calbiochem , La Jolla Ca) (50 ug/mL) with RNAase (400 U/mL) in 55~ v/v MK buffer (200mOsm) for 1-2 hours on ice. Cells were analyzed on a FACScan or Vantage flow cytometer (Becton Dickinson, San Jose, CA). Green fluorescence (CD4la-FITC) was collected along with linear and log signals for red fluorescence (PI) to determine DNA ploidy. All cells were collected to determine the percent of cells that were CD41+. Data analysis was performed using software by LYSIS (Becton Dickinson, San Jose, CA).
Percent of cells expressing the CD41 antigen was obtained from flow cytometry analysis(Percent).
Absolute (Abs) number of CD41+ cells/mL was calculated by: (Abs)=(Cell Count)*(Percent)/100.
2. Megakaryocyte fibrin clot assay.
CD34+ enriched population were isolated as described above. Cells were suspended at 25,000 cells/mL with or without cytokine(s) in a media consisting of a base Iscoves IMDM media supplemented with 0.3~ BSA, 0.4mg/mL apo-transferrin, 6.67uM FeCl2, 25ug/mL
CaCl2, 25ug/mL L-asparagine, 500ug/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-CD4la, 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 NcoI/HindIII DNA fragments from pMON30237 SEQ ID N0:53 and pMON30238 SEQ ID N0:54, containing the flt-3 (1-134) ligand and flt-3 (1-139) into vector, pMON30311 (derivative of pMON13058 - W095/21254), DNA digested with AfIIII/HindIII and SAP-treated (pMON30311 is a BHK-specific vector containing NcoI-IL-3 receptor agonist-IgG2B-AfIIII-HindIII as an insert) using standard ligation conditions. The ligation mixtures were used to transform competent DHSa 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 N0:21 and SEQ ID N0:22 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 N0:42 and SEQ ID
N0:43 respectively.
EXAMPLES 3 & 4 Construction of an expression vectors, pMON30247 and pMON30246, comprising a DNA sequence encoding a multi-functional receptor agonist comprising an IL-3 (15-125) variant joined via the IgG2b linker to a flt-3 (1-134) ligand and an IL-3 (15-125) variant joined via the GlySer linker to a flt-3 (1-134) ligand, respectively. Plasmids, pMON30246 and pMON30247, were constructed by cloning the gel purified NcoI/AfIIII restriction fragment from pMON30244 (GlySer linker) SEQ ID
N0:65 and pMON30245 (IgG2B linker) SEQ ID N0:66 respectively, into vector, pMON30237, digested with NcoI (which contains hFlt3L 1-134) as described in Examples 1 & 2. The DNA sequence of resulting genes, SEQ ID N0:13 and SEQ ID N0:14, encodes the proteins of SEQ ID N0:42 and SEQ ID
N0:43, 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 gei purified NcoI/AfIIII 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
N0:15 and SEQ ID N0:16, encodes the proteins of SEQ ID N0:36 and SEQ ID N0:37, 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 N0:29/134rev SEQ ID
N0:30 and N-term SEQ ID N0:29/139rev SEQ ID N0:31, 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 ~.1 dH20. 20 ~l of each purified PCR product were digested in 50 ~tl reaction mixture volumes with 10U each of Ncol and SnaBI for 90 minutes at 37°C. One ~,g of vector, pMON26431 (derivative of pMON13061 - W095/21254), was digested with 7.5U each of NcoI and SnaBI in a 20 ~1 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 N0:23 and SEQ
ID N0:24, encodes the proteins of SEQ ID N0:44 and SEQ ID N0:45, respectively.
r«..,...~ r., "
Construction of an expression vector, pMON30328, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-134) ligand joined via the IgG2b linker to a G-CSF receptor agonist. Plasmid, pMON30328, was constructed by subcloning the gel-purified NcoI/HindIII restriction fragment from pMON30237 into plasmid, pMON30309 (derivative of pMON13149 -W095/21254), digested with AfIIIT/HindIII
(contains G-CSF/IgG2b-AfIIII/HindIII) as described in Examples 1 & 2. The DNA sequence of resulting gene, SEQ ID N0:50, encodes the protein of SEQ ID
N0:60.

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. Piasmid, pMON30329, was constructed by subcloning the gel-purified NcoI/HindIII restriction fragment from pMON30238 into plasmid pMON30309 digested with AflIII/HindIII (which contains G-CSF/IgG2b-AfIIII/HindIII) as described in Examples 1 & 2.
The DNA sequence of resulting gene, SEQ ID N0:17, encodes the protein of SEQ ID N0:38.

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 N0:19, encodes the protein of SEQ ID
N0:40.

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 N0:58 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 N0:20, encodes the protein of SEQ ID N0:41.

Construction of an expression vector, pMON35767, comprising a DNA sequence encoding a multi-functional receptor agonist comprising a flt3 (1-139) ligand joined via the IgG2b linker to a G-CSF receptor agonist. Plasmid, pMON35767, was constructed by subcloning the gel-purified NcoI/HindIII restriction fragment from pMON32191 SEQ ID N0:20 into the BHK expression vector pMON3934, which is a derivative of pMON3359.
pMON3359 is a pUCl8-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 poly-adenylation (poly-A) signal which has been subcloned into the pUCl8 polylinker (See Hippenmeyer et al., Bio/Technology, 1993, pp.1037-1041). The DNA sequence of resulting gene, SEQ ID
N0:20, encodes the protein of SEQ ID N0:41.

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/Sacl restriction fragment from pMON32342 SEQ
ID N0:52 and the -290bp Sacl/SnaBI restriction fragment from pMON32393 into plasmid pMON30329 digested with Ncol/SnaBI as described in Examples 1 & 2. The DNA sequence of resulting gene, SEQ ID
N0:18, encodes the protein of SEQ ID N0:39.

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 N0:56, digested with NcoI/SnaBI as described in Examples 1 & 2. The DNA sequence of resulting gene, SEQ ID N0:25, encodes the protein of SEQ ID N0:46.

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 N0:26, encodes the protein of SEQ ID N0:47.

Construction of plasmid pMON46408 which encodes a mufti-functional receptor agonist comprising EPO
joined via a Gly Ser linker to a fit3 (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 AfIIII and HindIII. The following three DNA fragments were ligated together to form plasmid pMON46406:

WO 98/46750 PCT/US98/0'l5i 1 1. A 480 by 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 N0:33 which comprises a Stul-XmaI fragment encoding the terminal 6 amino acids of EPO and a portion of the GlySer polypeptide linker sequence 3. The 3,052 by NcoI-XmaI vector fragment of plasmid pMON13180 The ligation mixture was used to transform competent MON105 cells and transformants were selected on LB
Amp plates. Colonies were picked and analyzed by DNA
sequencing analysis to identify a correct clone. A
correct clone was assigned pMON46406.
In order to construct pMON46408, plasmid pMON46406 was digested with AflIII and HindIII and the vector portion was purified. This was ligated with the 423 by Ncol-HindIII fragment of plasmid pMON32342 SEQ ID N0:52, 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 N0:28, encodes the protein of SEQ ID N0:49.

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, w0 95/21197, w0 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: 11-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:1:
(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 (2) INFORMATION FOR SEQ ID N0:2:
(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 N0:2:
Gly Gly Gly Gly Ser (2) INFORMATION FOR SEQ ID N0:3:
(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 N0:3:
Gly Gly Gly Gly Gly Ser (2) INFORMATION FOR SEQ ID N0:4:
(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 N0:4:
Gly Ser (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 98/46750 ~ PGT/US98/07511 (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Ala Gly Ser (2) INFORMATION FOR SEQ ID N0:6:
(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 N0:6:
Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser (2) INFORMATION FOR SEQ ID N0:7:
(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 N0:7:
Ile Ser Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro (2) INFORMATION FOR SEQ ID N0:8:
(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 N0:8:
Ile Glu Gly Arg Ile Ser Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro (2) INFORMATION FOR SEQ ID N0:9:
(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 N0:9:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asg Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile 65 70 75 g0 His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (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 N0:10:
GCCACCCAGG ACTGCTCCTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCATGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACACC ATTGGGCCCT

GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTCTTTAG AGCAAGTGAG AAAGATCCAG

GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG

GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC

AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC

CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA

CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA

ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTC TGCTTTCCAG

CGCCGGGCAG GAGGGGTCCT GGTTGCTAGC CATCTGCAGA GCTTCCTGGA GGTGTCGTAC

CGCGTTCTAC GCCACCTTGC GCAGCCC

(2) INFORMATION FOR SEQ ID N0:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 349 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 98/4b750 67 PCT/US98/07511 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID N0:11:
Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gin Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 65 70 75 g0 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala VaI Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu (2) INFORMATION FOR SEQ ID N0:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1047 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG
CAAGTGAGAA 'AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC

AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT

CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT

AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG

GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG

CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC

TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC

TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTACGT AGAGGGCGGT

GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT

AAAGAATCTC ATAAATCTCC AAACATGGCT ACCCAGGACT GCTCCTTCCA ACACAGCCCC

ATCTCCTCCG ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCT TCAAGATTAC

CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGGCCT CTGGCGGCTG

GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGGTCCAA GATGCAAGGC

TTGCTGGAGC GCGTGAACAC GGAGATACAC TTTGTCACCA AATGTGCCTT TCAGCCCCCC

CCCAGCTGTC TTCGCTTCGT CCAGACCAAC ATCTCCCGCC TCCTGCAGGA GACCTCCGAG

CAGCTGGTGG CGCTGAAGCC CTGGATCACT CGCCAGAACT TCTCCCGGTG CCTGGAGCTG

CAGTGTCAGC CCGACTCCTC AACCCTG

(2) INFORMATION FOR SEQ ID N0:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 798 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT
TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT

CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT

ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT

CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC

TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC

CCGGGTGGTG GTTCTGGCGG CGGCTCCAAC ATGGCCACCC AGGACTGCTC CTTCCAACAC

AGCCCCATCT CCTCCGACTT CGCTGTCAAA ATCCGTGAGC TGTCTGACTA CCTGCTTCAA

GATTACCCAG TCACCGTGGC CTCCAACCTG CAGGACGAGG AGCTCTGCGG GGCGCTCTGG

CGGCTGGTCC TGGCACAGCG CTGGATGGAG CGGCTCAAGA CTGTCGCTGG GTCCAAGATG

CAAGGCTTGC TGGAGCGCGT GAACACGGAG ATACACTTTG TCACCAAATG TGCCTTTCAG

CCCCCCCCCA GCTGTCTTCG CTTCGTCCAG ACCAACATCT CCCGCCTCCT GCAGGAGACC

TCCGAGCAGC TGGTGGCGCT GAAGCCCTGG ATCACTCGCC AGAACTTCTC CCGGTGCCTG

GAGCTGCAGT GTCAGCCC

(2) INFORMATION FOR SEQ ID N0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 843 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:14:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT
TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT

CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT

ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT

CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC

TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC

CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT

CATAAATCTC CAAACATGGC CACCCAGGAC TGCTCCTTCC AACACAGCCC CATCTCCTCC

GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC

GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGCGC TCTGGCGGCT GGTCCTGGCA

CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG

CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT

CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG

WO 98/46750 ~0 PCT/US98/07511 GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG

CCC

(2) INFORMATION FOR SEQ ID N0:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 813 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:15:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT
TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT

CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT

ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT

CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC

TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC

CCGGGTGGTG GTTCTGGCGG CGGCTCCAAC ATGGCCACCC AGGACTGCTC CTTCCAACAC

AGCCCCATCT CCTCCGACTT CGCTGTCAAA ATCCGTGAGC TGTCTGACTA CCTGCTTCAA

GATTACCCAG TCACCGTGGC CTCCAACCTG CAGGACGAGG AGCTCTGCGG GGCGCTCTGG

CGGCTGGTCC TGGCACAGCG CTGGATGGAG CGGCTCAAGA CTGTCGCTGG GTCCAAGATG

CAAGGCTTGC TGGAGCGCGT GAACACGGAG ATACACTTTG TCACCAAATG TGCCTTTCAG

CCCCCCCCCA GCTGTCTTCG CTTCGTCCAG ACCAACATCT CCCGCCTCCT GCAGGAGACC

TCCGAGCAGC TGGTGGCGCT GAAGCCCTGG ATCACTCGCC AGAACTTCTC CCGGTGCCTG

GAGCTGCAGT GTCAGCCCGA CTCCTCAACC CTG

(2) INFORMATION FOR SEQ ID N0:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 858 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:16:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTGCACCT

WO 98/46750 ~ 1 PCT/US98/07511 TTGCTGGACC CGAACAACCT CAATGACGAA GACGTCTCTA TCCTGATGGA CCGAAACCTT

CGACTTCCAA ACCTGGAGAG CTTCGTAAGG GCTGTCAAGA ACTTAGAAAA TGCATCAGGT

ATTGAGGCAA'TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT

CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC

TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC

CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT

CATAAATCTC CAAACATGGC CACCCAGGAC TGCTCCTTCC AACACAGCCC CATCTCCTCC

GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC

GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGCGC TCTGGCGGCT GGTCCTGGCA

CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG

CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT

CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG

GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG

CCCGACTCCT CAACCCTG

(2) INFORMATION FOR SEQ ID N0:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1047 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:17:
GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG
CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC

AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT

CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT

AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG

GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG

CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC

TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC

TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTACGT AGAGGGCGGT

WO 98/46750 ~2 PCT/US98/07511 GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT

AAAGAATCTC ATAAATCTCC AAACATGGCC ACCCAGGACT GCTCCTTCCA ACACAGCCCC

ATCTCCTCCG 'ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCT TCAAGATTAC

CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGGCCT CTGGCGGCTG

GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGGTCCAA GATGCAAGGC

TTGCTGGAGC GCGTGAACAC GGAGATACAC TTTGTCACCA AATGTGCCTT TCAGCCCCCC

CCCAGCTGTC TTCGCTTCGT CCAGACCAAC ATCTCCCGCC TCCTGCAGGA GACCTCCGAG

CAGCTGGTGG CGCTGAAGCC CTGGATCACT CGCCAGAACT TCTCCCGGTG CCTGGAGCTG

CAGTGTCAGC CCGACTCCTC AACCCTG

(2) INFORMATION FOR SEQ ID N0:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 942 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:18:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACCCA GGACTGCTCC

TTCCAACACA GCCCCATCTC CTCCGACTTC GCTGTCAAAA TCCGTGAGCT GTCTGACTAC

CTGCTTCAAG ATTACCCAGT CACCGTGGCC TCCAACCTGC AGGACGAGGA GCTCTGCGGG

GGCCTCTGGC GGCTGGTCCT GGCACAGCGC TGGATGGAGC GGCTCAAGAC TGTCGCTGGG

TCCAAGATGC AAGGCTTGCT GGAGCGCGTG AACACGGAGA TACACTTTGT CACCAAATGT

GCCTTTCAGC CCCCCCCCAG CTGTCTTCGC TTCGTCCAGA CCAACATCTC CCGCCTCCTG

CAGGAGACCT CCGAGCAGCT GGTGGCGCTG AAGCCCTGGA TCACTCGCCA GAACTTCTCC

CGGTGCCTGG AGCTGCAGTG TCAGCCCGAC TCCTCAACCC TG

(2) INFORMATION FOR SEQ ID N0:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1047 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:19:
GCCACCCAGG ACTGCTCCTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCATGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACACC ATTAGGCCCT

GCCAGCTCCC TGCCCCAGAG CTTCCTGCTC AAGTGCTTAG AGCAAGTGAG GAAGATCCAG

GGCGATGGCG CAGCGCTCCA GGAGAAGCTG TGTGCCACCT ACAAGCTGTG CCACCCCGAG

GAGCTGGTGC TGCTCGGACA CTCTCTGGGC ATCCCCTGGG CTCCCCTGAG CTCCTGCCCC

AGCCAGGCCC TGCAGCTGGC AGGCTGCTTG AGCCAACTCC ATAGCGGCCT TTTCCTCTAC

CAGGGGCTCC TGCAGGCCCT GGAAGGGATA TCCCCCGAGT TGGGTCCCAC CTTGGACACA

CTGCAGCTGG ACGTCGCCGA CTTTGCCACC ACCATCTGGC AGCAGATGGA AGAACTGGGA

ATGGCCCCTG CCCTGCAGCC CACCCAGGGT GCCATGCCGG CCTTCGCCTC TGCTTTCCAG

CGCCGGGCAG GAGGGGTCCT GGTTGCTAGC CATCTGCAGA GCTTCCTGGA GGTGTCGTAC

CGCGTTTTAC GCCACCTTGC GCAGCCC

(2) INFORMATION FOR SEQ ID N0:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1003 base pairs (B) TYPE: nucleic acid WO 98/46750 ~4 PCT/US98/07511 (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi)'SEQUENCE DESCRIPTION: SEQ ID N0:20:
GGCCACTCAG GACTGCTCTT TTCAACACAG CCCCATCTCC TCCGACTTCG CTGTCAAAAT
CCGTGAGCTG TCTGACTACC TGCTTCAAGA TTACCCAGTC ACCGTGGCCT CCAACCTGCA

GGACGAGGAG CTCTGCGGGG GCCTCTGGCG GCTGGTCCTG GCACAGCGCT GGATGGAGCG

GCTCAAGACT GTCGCTGGGT CCAAGATGCA AGGCTTGCTG GAGCGCGTGA ACACGGAGAT

ACACTTTGTC ACCAAATGTG CCTTTCAGCC CCCCCCCAGC TGTCTTCGCT TCGTCCAGAC

CAACATCTCC CGCCTCCTGC AGGAGACCTC CGAGCAGCTG GTGGCGCTGA AGCCCTGGAT

CACTCGCCAG AACTTCTCCC GGTGCCTGGA GCTGCAGTGT CAGCCCGACT CCTCAACCCT

GTACGTAGAG GGCGGTGGAG GCTCCCCGGG TGGTGGTTCT GGCGGCGGCT CCAACATGGC

TACACCATTG GGCCCTGCCA GCTCCCTGCC CCAGAGCTTC CTGCTCAAGT CTTTAGAGCA

AGTGAGAAAG ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA

GCTGTGCCAC CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC CCTGGGCTCC

CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC TGCTTGAGCC AACTCCATAG

CGGCCTTTTC CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCC CCGAGTTGGG

TCCCACCTTG GACACACTGC AGCTGGACGT CGCCGACTTT GCCACCACCA TCTGGCAGCA

GATGGAAGAA CTGGGAATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT

CGCCTCTGCT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT

CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCG

(2) INFORMATION FOR SEQ ID N0:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 843 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:21:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTAACCCT
TTGCTGGACC CGAACAACCT CAATTCTGAA GACATGGATA TCCTGATGGA ACGAAACCTT

CGAACTCCAA ACCTGCTCGC ATTCGTAAGG GCTGTCAAGC ACTTAGAAAA TGCATCAGGT

WO 98/46750 ~5 PGT/US98/07511 ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT

CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC

TATCTGGTTA'CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC

CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT

CATAAATCTC CAAACATGGC GACTCAGGAC TGTTCTTTCC AACACAGCCC CATCTCCTCC

GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC

GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGGCC TCTGGCGGCT GGTCCTGGCA

CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG

CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT

CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG

GCGCTGAAGC CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG

CCC

(2) INFORMATION FOR SEQ ID N0:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 858 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:22:
GCTAACTGCT CTATAATGAT CGATGAAATT ATACATCACT TAAAGAGACC ACCTAACCCT
TTGCTGGACC CGAACAACCT CAATTCTGAA GACATGGATA TCCTGATGGA ACGAAACCTT

CGAACTCCAA ACCTGCTCGC ATTCGTAAGG GCTGTCAAGC ACTTAGAAAA TGCATCAGGT

ATTGAGGCAA TTCTTCGTAA TCTCCAACCA TGTCTGCCCT CTGCCACGGC CGCACCCTCT

CGACATCCAA TCATCATCAA GGCAGGTGAC TGGCAAGAAT TCCGGGAAAA ACTGACGTTC

TATCTGGTTA CCCTTGAGCA AGCGCAGGAA CAACAGTACG TAGAGGGCGG TGGAGGCTCC

CCGGGTGAAC CGTCTGGTCC AATCTCTACT ATCAACCCGT CTCCTCCGTC TAAAGAATCT

CATAAATCTC CAAACATGGC AACCCAGGAC TGCTCTTTTC AACACAGCCC CATCTCCTCC

GACTTCGCTG TCAAAATCCG TGAGCTGTCT GACTACCTGC TTCAAGATTA CCCAGTCACC

GTGGCCTCCA ACCTGCAGGA CGAGGAGCTC TGCGGGGGCC TCTGGCGGCT GGTCCTGGCA

CAGCGCTGGA TGGAGCGGCT CAAGACTGTC GCTGGGTCCA AGATGCAAGG CTTGCTGGAG

WO 98/46750 ~6 PCT/US98/07511 CGCGTGAACA CGGAGATACA CTTTGTCACC AAATGTGCCT TTCAGCCCCC CCCCAGCTGT

CTTCGCTTCG TCCAGACCAA CATCTCCCGC CTCCTGCAGG AGACCTCCGA GCAGCTGGTG

GCGCTGAAGC 'CCTGGATCAC TCGCCAGAAC TTCTCCCGGT GCCTGGAGCT GCAGTGTCAG

CCCGACTCCT CAACCCTG

(2) INFORMATION FOR SEQ ID N0:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 843 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:23:
GCCACTCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCTACGT AGAGGGCGGT

GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT

AAAGAATCTC ATAAATCTCC AAACATGGCT AACTGCTCTA TAATGATCGA TGAAATTATA

CATCACTTAA AGAGACCACC TAACCCTTTG CTGGACCCGA ACAACCTCAA TTCTGAAGAC

ATGGATATCC TGATGGAACG AAACCTTCGA ACTCCAAACC TGCTCGCATT CGTAAGGGCT

GTCAAGCACT TAGAAAATGC ATCAGGTATT GAGGCAATTC TTCGTAATCT CCAACCATGT

CTGCCCTCTG CCACGGCCGC ACCCTCTCGA CATCCAATCA TCATCAAGGC AGGTGACTGG

CAAGAATTCC GGGAAAAACT GACGTTCTAT CTGGTTACCC TTGAGCAAGC GCAGGAACAA

CAG

(2) INFORMATION FOR SEQ ID N0:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 858 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear WO 98/46750 ~~ PGT/US98/07511 (xi) SEQUENCE DESCRIPTION: SEQ ID N0:24:
GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTAACTG CTCTATAATG

ATCGATGAAA TTATACATCA CTTAAAGAGA CCACCTAACC CTTTGCTGGA CCCGAACAAC

CTCAATTCTG AAGACATGGA TATCCTGATG GAACGAAACC TTCGAACTCC AAACCTGCTC

GCATTCGTAA GGGCTGTCAA GCACTTAGAA AATGCATCAG GTATTGAGGC AATTCTTCGT

AATCTCCAAC CATGTCTGCC CTCTGCCACG GCCGCACCCT CTCGACATCC AATCATCATC

AAGGCAGGTG ACTGGCAAGA ATTCCGGGAA AAACTGACGT TCTATCTGGT TACCCTTGAG

CAAGCGCAGG AACAACAG

(2) INFORMATION FOR SEQ ID N0:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 939 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:25:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC

WO 98/46750 7g PCT/ITS98/07511 ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GGTGGTTCTG GCGGCGGCTC CAACATGGCG

TCTCCGGCGC'CGCCTGCTTG TGACCTCCGA GTCCTCAGTA AACTGCTTCG TGACTCCCAT

GTCCTTCACA GCAGACTGAG CCAGTGCCCA GAGGTTCACC CTTTGCCTAC ACCTGTCCTG

CTGCCTGCTG TGGACTTTAG CTTGGGAGAA TGGAAAACCC AGATGGAGGA GACCAAGGCA

CAGGACATTC TGGGAGCAGT GACCCTTCTG CTGGAGGGAG TGATGGCAGC ACGGGGACAA

CTGGGACCCA CTTGCCTCTC ATCCCTCCTG GGGCAGCTTT CTGGACAGGT CCGTCTCCTC

CTTGGGGCCC TGCAGAGCCT CCTTGGAACC CAGCTTCCTC CACAGGGCAG GACCACAGCT

CACAAGGATC CCAATGCCAT CTTCCTGAGC TTCCAACACC TGCTCCGAGG AAAGGTGCGT

TTCCTGATGC TTGTAGGAGG GTCCACCCTC TGCGTCAGG

(2) INFORMATION FOR SEQ ID N0:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 996 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:26:
GCGTCCCCAG CTCCACCTGC TTGTGACCTC CGAGTCCTCA GTAAACTGCT TCGTGACTCC
CATGTCCTTC ACAGCAGACT GAGCCAGTGC CCAGAGGTTC ACCCTTTGCC TACACCTGTC

CTGCTGCCTG CTGTGGACTT TAGCTTGGGA GAATGGAAAA CCCAGATGGA GGAGACCAAG

GCACAGGACA TTCTGGGAGC AGTGACCCTT CTGCTGGAGG GAGTGATGGC AGCACGGGGA

CAACTGGGAC CCACTTGCCT CTCATCCCTC CTGGGGCAGC TTTCTGGACA GGTCCGTCTC

CTCCTTGGGG CCCTGCAGAG CCTCCTTGGA ACCCAGCTTC CTCCACAGGG CAGGACCACA

GCTCACAAGG ATCCCAATGC CATCTTCCTG AGCTTCCAAC ACCTGCTCCG AGGAAAGGTG

CGTTTCCTGA TGCTTGTAGG AGGGTCCACC CTCTGCGTCA GGGAATTCCA TGCATACGTA

GAGGGCGGTG GAGGCTCCCC GGGTGAACCG TCTGGTCCAA TCTCTACTAT CAACCCGTCT

CCTCCGTCTA AAGAATCTCA TAAATCTCCA AACATGGCTA CCCAGGACTG CTCCTTCCAA

CACAGCCCCA TCTCCTCCGA CTTCGCTGTC AAAATCCGTG AGCTGTCTGA CTACCTGCTT

CAAGATTACC CAGTCACCGT GGCCTCCAAC CTGCAGGACG AGGAGCTCTG CGGGGGCCTC

TGGCGGCTGG TCCTGGCACA GCGCTGGATG GAGCGGCTCA AGACTGTCGC TGGGTCCAAG

WO 98/46750 ~9 PCT/US98/07511 ATGCAAGGCT TGCTGGAGCG CGTGAACACG GAGATACACT TTGTCACCAA ATGTGCCTTT

CAGCCCCCCC CCAGCTGTCT TCGCTTCGTC CAGACCAACA TCTCCCGCCT CCTGCAGGAG

ACCTCCGAGC'AGCTGGTGGC GCTGAAGCCC TGGATCACTC GCCAGAACTT CTCCCGGTGC

CTGGAGCTGC AGTGTCAGCC CGACTCCTCA ACCCTG

(2) INFORMATION FOR SEQ ID N0:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1020 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:27:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCCCCACC ACGCCTCATC

TGTGACAGCC GAGTCCTGGA GAGGTACCTC ~'TGGAGGCCA AGGAGGCCGA GAATATCACG

ACGGGCTGTG CTGAACACTG CAGCTTGAAT GAGAATATCA CTGTCCCAGA CACCAAAGTT

AATTTCTATG CCTGGAAGAG GATGGAGGTC GGGCAGCAGG CCGTAGAAGT CTGGCAGGGC

CTGGCCCTGC TGTCGGAAGC TGTCCTGCGG GGCCAGGCCC TGTTGGTCAA CTCTTCCCAG
?80 CCGTGGGAGC CCCTGCAGCT GCATGTGGAT AAAGCCGTCA GTGGCCTTCG CAGCCTCACC

ACTCTGCTTC GGGCTCTGCG AGCCCAGAAG GAAGCCATCT CCCCTCCAGA TGCGGCCTCA

GCTGCTCCAC TCCGAACAAT CACTGCTGAC ACTTTCCGCA AACTCTTCCG AGTCTACTCC

AATTTCCTCC GGGGAAAGCT GAAGCTGTAC ACAGGGGAGG CCTGCAGGAC AGGGGACAGA

(2) INFORMATION FOR SEQ ID N0:28:
(i) SEQUENCE CHARACTERISTICS:

WO 98/46750 g0 PGT/US98/07511 (A) LENGTH: 975 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:28:
GCCCCACCAC GCCTCATCTG TGACAGCCGA GTCCTGGAGA GGTACCTCTT GGAGGCCAAG
GAGGCCGAGA ATATCACGAC GGGCTGTGCT GAACACTGCA GCTTGAATGA GAATATCACT

GTCCCAGACA CCAAAGTTAA TTTCTATGCC TGGAAGAGGA TGGAGGTCGG GCAGCAGGCC

GTAGAAGTCT GGCAGGGCCT GGCCCTGCTG TCGGAAGCTG TCCTGCGGGG CCAGGCCCTG

TTGGTCAACT CTTCCCAGCC GTGGGAGCCC CTGCAGCTGC ATGTGGATAA AGCCGTCAGT

GGCCTTCGCA GCCTCACCAC TCTGCTTCGG GCTCTGCGAG CCCAGAAGGA AGCCATCTCC

CCTCCAGATG CGGCCTCAGC TGCTCCACTC CGAACAATCA CTGCTGACAC TTTCCGCAAA

CTCTTCCGAG TCTACTCCAA TTTCCTCCGG GGAAAGCTGA AGCTGTACAC AGGGGAGGCC

TGCAGGACAG GGGACAGATA CGTAGAGGGC GGTGGAGGCT CCCCGGGTGG TGGTTCTGGC

GGCGGCTCCA ACATGGCCAC TCAGGACTGC TCTTTTCAAC ACAGCCCCAT CTCCTCCGAC

TTCGCTGTCA AAATCCGTGA GCTGTCTGAC TACCTGCTTC AAGATTACCC AGTCACCGTG

GCCTCCAACC TGCAGGACGA GGAGCTCTGC GGGGGCCTCT GGCGGCTGGT CCTGGCACAG

CGCTGGATGG AGCGGCTCAA GACTGTCGCT GGGTCCAAGA TGCAAGGCTT GCTGGAGCGC

GTGAACACGG AGATACACTT TGTCACCAAA TGTGCCTTTC AGCCCCCCCC CAGCTGTCTT

CGCTTCGTCC AGACCAACAT CTCCCGCCTC CTGCAGGAGA CCTCCGAGCA GCTGGTGGCG

CTGAAGCCCT GGATCACTCG CCAGAACTTC TCCCGGTGCC TGGAGCTGCA GTGTCAGCCC

GACTCCTCAA CCCTG

(2) INFORMATION FOR SEQ ID N0:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:29:
GTAGTCCATG GCCACCCAGG ACTGCTCC

(2) INFORMATION FOR SEQ ID N0:30:

wo 9s~~w~so 81 rcrms9sro~si ~
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:30:
GCATTACGTA GGGCTGACAC TGCAGCTCCA G

(2) INFORMATION FOR SEQ ID N0:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:31:
GCATTACGTA CAGGGTTGAG GAGTCGGGCT G

(2) INFORMATION FOR SEQ ID N0:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:32:
CCTGCAGGAC AGGGGACAGA TACGTAGAGG GCGGTGGAGG CTCC

(2) INFORMATION FOR SEQ ID N0:33:
(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 N0:33:
CCGGGGAGCC TCCACCGCCC TCTACGTATC TGTCCCCTGT CCTGCAGG

(2) INFORMATION FOR SEQ ID N0:34:

WO 98/46750 82 PC'T/US98/07511 (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 N0:34:
Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly Ser Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Ala Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro (2) INFORMATION FOR SEQ ID N0:35:
(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 N0:35:
Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe 35~ 40 45 Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly 180 i85 190 Ala Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro (2) INFORMATION FOR SEQ ID N0:36:
(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 N0:36:
Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser 65 70 75 g0 WO 98/46750 g4 PCT/US98/07511 Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln Tyr Val Glti Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly Ser Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Ala Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu (2) INFORMATION FOR SEQ ID N0:37:
(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 N0:37:
Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Ala Pro Leu Leu Asp Pro Asn Asn Leu Asn Asp Glu Asp Val Ser Ile Leu Met Asp Arg Asn Leu Arg Leu Pro Asn Leu Glu Ser Phe Val Arg Ala Val Lys Asn Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Ala Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu {2) INFORMATION FOR SEQ ID N0:38:
(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 N0:38:
Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Giy Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr WO 98146750 8b PCT/US98/0'7511 Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln~Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu (2) INFORMATION FOR SEQ ID N0:39:
(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 N0:39:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu (2) INFORMATION FOR SEQ ID N0:40:
(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 N0:40:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (2) INFORMATION FOR SEQ ID N0:41:
(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 N0:41:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly Ser Asn Met Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (2} INFORMATION FOR SEQ ID N0:42:
(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 N0:42:
Ala Asn Cys Ser IIe Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro (2) INFORMATION FOR SEQ ID N0:43:
(i) SEQUENCE CHARACTERISTICS:

WO 98/~t6750 90 PCT/US98/07511 (A) LENGTH: 286 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID N0:43:
Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Vai Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu (2) INFORMATION FOR SEQ ID N0:44:
(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 N0:44:

Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln (2) INFORMATION FOR SEQ ID N0:45:
(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 N0:45:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Asn Cys Ser Ile Met Ile Asp Glu Ile Ile His His Leu Lys Arg Pro Pro Asn Pro Leu Leu Asp Pro Asn Asn Leu Asn Ser Glu Asp Met Asp Ile Leu Met Glu Arg Asn Leu Arg Thr Pro Asn Leu Leu Ala Phe Val Arg Ala Val Lys His Leu Glu Asn Ala Ser Gly Ile Glu Ala Ile Leu Arg Asn Leu Gln Pro Cys Leu Pro Ser Ala Thr Ala Ala Pro Ser Arg His Pro Ile Ile Ile Lys Ala Gly Asp Trp Gln Glu Phe Arg Glu Lys Leu Thr Phe Tyr Leu Val Thr Leu Glu Gln Ala Gln Glu Gln Gln (2) INFORMATION FOR SEQ ID N0:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 313 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: None (xi) SEQUENCE DESCRIPTION: SEQ ID N0:46:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu VaI Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly Ser Asn Met Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg (2) INFORMATION FOR SEQ ID N0:47:
(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 N0:47:
Ala Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Glu Phe His Ala Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro 210 ~ 215 220 Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu (2) INFORMATION FOR SEQ ID N0:48:
(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 N0:48:
Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu GIu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg (2) INFORMATION FOR SEQ ID N0:49:
(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 N0:49:
Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu Thr Thr Leu Leu Arg Ala Leu Arg Ala Gln Lys Glu Ala Ile Ser Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp Arg Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly Ser Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu (2) INFORMATION FOR SEQ ID N0:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1032 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:50:
GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG
CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC

AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT

CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT

AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG

GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG

CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC

TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC

TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCCTACGT AGAGGGCGGT

GGAGGCTCCC CGGGTGAACC GTCTGGTCCA ATCTCTACTA TCAACCCGTC TCCTCCGTCT

AAAGAATCTC ATAAATCTCC AAACATGGCC ACCCAGGACT GCTCCTTCCA ACACAGCCCC

ATCTCCTCCG ACTTCGCTGT CAAAATCCGT GAGCTGTCTG ACTACCTGCT TCAAGATTAC

CCAGTCACCG TGGCCTCCAA CCTGCAGGAC GAGGAGCTCT GCGGGGGCCT CTGGCGGCTG

GTCCTGGCAC AGCGCTGGAT GGAGCGGCTC AAGACTGTCG CTGGGTCCAA GATGCAAGGC

TTGCTGGAGC GCGTGAACAC GGAGATACAC TTTGTCACCA AATGTGCCTT TCAGCCCCCC

CCCAGCTGTC TTCGCTTCGT CCAGACCAAC ATCTCCCGCC TCCTGCAGGA GACCTCCGAG

CAGCTGGTGG CGCTGAAGCC CTGGATCACT CGCCAGAACT TCTCCCGGTG CCTGGAGCTG

CAGTGTCAGC ' CC

(2) INFORMATION FOR SEQ ID N0:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1005 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:51:
ATGGCCACTC AGGACTGCTC TTTTCAACAC AGCCCCATCT CCTCCGACTT CGCTGTCAAA
ATCCGTGAGC TGTCTGACTA CCTGCTTCAA GATTACCCAG TCACCGTGGC CTCCAACCTG

CAGGACGAGG AGCTCTGCGG GGGCCTCTGG CGGCTGGTCC TGGCACAGCG CTGGATGGAG

CGGCTCAAGA CTGTCGCTGG GTCCAAGATG CAAGGCTTGC TGGAGCGCGT GAACACGGAG

ATACACTTTG TCACCAAATG TGCCTTTCAG CCCCCCCCCA GCTGTCTTCG CTTCGTCCAG

ACCAACATCT CCCGCCTCCT GCAGGAGACC TCCGAGCAGC TGGTGGCGCT GAAGCCCTGG

ATCACTCGCC AGAACTTCTC CCGGTGCCTG GAGCTGCAGT GTCAGCCCGA CTCCTCAACC

CTGTACGTAG AGGGCGGTGG AGGCTCCCCG GGTGGTGGTT CTGGCGGCGG CTCCAACATG

GCTACACCAT TGGGCCCTGC CAGCTCCCTG CCCCAGAGCT TCCTGCTCAA GTCTTTAGAG

CAAGTGAGAA AGATCCAGGG CGATGGCGCA GCGCTCCAGG AGAAGCTGTG TGCCACCTAC

AAGCTGTGCC ACCCCGAGGA GCTGGTGCTG CTCGGACACT CTCTGGGCAT CCCCTGGGCT

CCCCTGAGCT CCTGCCCCAG CCAGGCCCTG CAGCTGGCAG GCTGCTTGAG CCAACTCCAT

AGCGGCCTTT TCCTCTACCA GGGGCTCCTG CAGGCCCTGG AAGGGATATC CCCCGAGTTG

GGTCCCACCT TGGACACACT GCAGCTGGAC GTCGCCGACT TTGCCACCAC CATCTGGCAG

CAGATGGAAG AACTGGGAAT GGCCCCTGCC CTGCAGCCCA CCCAGGGTGC CATGCCGGCC

TTCGCCTCTG CTTTCCAGCG CCGGGCAGGA GGGGTCCTGG TTGCTAGCCA TCTGCAGAGC

TTCCTGGAGG TGTCGTACCG CGTTCTACGC CACCTTGCGC AGCCG

(2) INFORMATION FOR SEQ ID N0:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 420 base pairs (B) TYPE: nucleic acid I

WO 98146750 9g PGT/US98/07511 (C) STRANDEDNESS: single (D} TOPOLOGY: linear {xi)'SEQUENCE DESCRIPTION: SEQ ID N0:52:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG

(2) INFORMATION FOR SEQ ID N0:53:
{i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 405 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:53:
GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGC GCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCC

(2) INFORMATION FOR SEQ ID N0:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 420 base pairs (B} TYPE: nucleic acid (C} STRANDEDNESS: single {D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:54:

GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC'TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG

(2) INFORMATION FOR SEQ ID N0:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 420 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:55:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG

(2) INFORMATION FOR SEQ ID N0:56:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 942 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:56:
GCCACTCAGG ACTGCTCTTT TCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

WO 98/46750 i~~ PCTNS98/0'1511 CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC~GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTACCCA GGACTGCTCC

TTCCAACACA GCCCCATCTC CTCCGACTTC GCTGTCAAAA TCCGTGAGCT GTCTGACTAC

CTGCTTCAAG ATTACCCAGT CACCGTGGCC TCCAACCTGC AGGACGAGGA GCTCTGCGGG

GGCCTCTGGC GGCTGGTCCT GGCACAGCGC TGGATGGAGC GGCTCAAGAC TGTCGCTGGG

TCCAAGATGC AAGGCTTGCT GGAGCGCGTG AACACGGAGA TACACTTTGT CACCAAATGT

GCCTTTCAGC CCCCCCCCAG CTGTCTTCGC TTCGTCCAGA CCAACATCTC CCGCCTCCTG

CAGGAGACCT CCGAGCAGCT GGTGGCGCTG AAGCCCTGGA TCACTCGCCA GAACTTCTCC

CGGTGCCTGG AGCTGCAGTG TCAGCCCGAC TCCTCAACCC TG

(2) INFORMATION FOR SEQ ID N0:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1003 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:57:
GGCCACTCAG GACTGCTCTT TTCAACACAG CCCCATCTCC TCCGACTTCG CTGTCAAAAT
CCGTGAGCTG TCTGACTACC TGCTTCAAGA TTACCCAGTC ACCGTGGCCT CCAACCTGCA

GGACGAGGAG CTCTGCGGGG GCCTCTGGCG GCTGGTCCTG GCACAGCGCT GGATGGAGCG

GCTCAAGACT GTCGCTGGGT CCAAGATGCA AGGCTTGCTG GAGCGCGTGA ACACGGAGAT

ACACTTTGTC ACCAAATGTG CCTTTCAGCC CCCCCCCAGC TGTCTTCGCT TCGTCCAGAC

CAACATCTCC CGCCTCCTGC AGGAGACCTC CGAGCAGCTG GTGGCGCTGA AGCCCTGGAT

CACTCGCCAG AACTTCTCCC GGTGCCTGGA GCTGCAGTGT CAGCCCGACT CCTCAACCCT

GTACGTAGAG GGCGGTGGAG GCTCCCCGGG TGGTGGTTCT GGCGGCGGCT CCAACATGGC

TACACCATTG GGCCCTGCCA GCTCCCTGCC CCAGAGCTTC CTGCTCAAGT CTTTAGAGCA

AGTGAGAAAG ATCCAGGGCG ATGGCGCAGC GCTCCAGGAG AAGCTGTGTG CCACCTACAA

WO 98/46750 ifl~ PCT/US98/07511 GCTGTGCCAC CCCGAGGAGC TGGTGCTGCT CGGACACTCT CTGGGCATCC CCTGGGCTCC

CCTGAGCTCC TGCCCCAGCC AGGCCCTGCA GCTGGCAGGC TGCTTGAGCC AACTCCATAG

CGGCCTTTTC'CTCTACCAGG GGCTCCTGCA GGCCCTGGAA GGGATATCCC CCGAGTTGGG

TCCCACCTTG GACACACTGC AGCTGGACGT CGCCGACTTT GCCACCACCA TCTGGCAGCA

GATGGAAGAA CTGGGAATGG CCCCTGCCCT GCAGCCCACC CAGGGTGCCA TGCCGGCCTT

CGCCTCTGCT TTCCAGCGCC GGGCAGGAGG GGTCCTGGTT GCTAGCCATC TGCAGAGCTT

CCTGGAGGTG TCGTACCGCG TTCTACGCCA CCTTGCGCAG CCG

(2) INFORMATION FOR SEQ ID N0:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 858 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:58:
GCCACCCAGG ACTGCTCCTT CCAACACAGC CCCATCTCCT CCGACTTCGC TGTCAAAATC
CGTGAGCTGT CTGACTACCT GCTTCAAGAT TACCCAGTCA CCGTGGCCTC CAACCTGCAG

GACGAGGAGC TCTGCGGGGG CCTCTGGCGG CTGGTCCTGG CACAGCGCTG GATGGAGCGG

CTCAAGACTG TCGCTGGGTC CAAGATGCAA GGCTTGCTGG AGCGCGTGAA CACGGAGATA

CACTTTGTCA CCAAATGTGC CTTTCAGCCC CCCCCCAGCT GTCTTCGCTT CGTCCAGACC

AACATCTCCC GCCTCCTGCA GGAGACCTCC GAGCAGCTGG TGGCGCTGAA GCCCTGGATC

ACTCGCCAGA ACTTCTCCCG GTGCCTGGAG CTGCAGTGTC AGCCCGACTC CTCAACCCTG

TACGTAGAGG GCGGTGGAGG CTCCCCGGGT GAACCGTCTG GTCCAATCTC TACTATCAAC

CCGTCTCCTC CGTCTAAAGA ATCTCATAAA TCTCCAAACA TGGCTAACTG CTCTATAATG

ATCGATGAAA TTATACATCA CTTAAAGAGA CCACCTAACC CTTTGCTGGA CCCGAACAAC

CTCAATTCTG AAGACATGGA TATCCTGATG GAACGAAACC TTCGAACTCC AAACCTGCTC

GCATTCGTAA GGGCTGTCAA GCACTTAGAA AATGCATCAG GTATTGAGGC AATTCTTCGT

AATCTCCAAC CATGTCTGCC CTCTGCCACG GCCGCACCCT CTCGACATCC AATCATCATC

AAGGCAGGTG ACTGGCAAGA ATTCCGGGAA AAACTGACGT TCTATCTGGT TACCCTTGAG

CAAGCGCAGG AACAACAG

(2) INFORMATION FOR SEQ ID N0:59:

(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 N0:59:
ATGGCTCCAA TGACTCAGAC TACTTCTCTT AAGACTTCTT GGGTTAACTG CTCTAACATG
ATCGATGAAA TTATAACACA CTTAAAGCAG CCACCTTTGC CTTTGCTGGA CTTCAACAAC

CTCAATGGGG AAGACCAAGA CATTCTGATG GAAAATAACC TTCGAAGGCC AAACCTGGAG

GCATTCAACA GGGCTGTCAA GAGTTTACAG AATGCATCAG CAATTGAGAG CATTCTTAAA

AATCTCCTGC CATGTCTGCC CCTGGCCACG GCCGCACCCA CGCGACATCC AATCCATATC

AAGGACGGTG ACTGGAATGA ATTCCGTCGT AAACTGACCT TCTATCTGAA AACCTTGGAG

AACGCGCAGG CTCAACAGAC CACTCTGTCG CTAGCGATCT TT

(2) INFORMATION FOR SEQ ID N0:60:
(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 N0:60:
Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Ser Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro IIe Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gin Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro (2) INFORMATION FOR SEQ ID N0:61:
(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 N0:61:
Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile 85 90 ~ 95 His Ile Lys Asp Gly Asp Trp Asn Gly Ile Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe (2) INFORMATION FOR SEQ ID N0:62:

WO 98/46750 1~ PCT/US98/07511 (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 N0:62:
Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Gly Ile Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Gly Gly Ser Gly Gly Gly Ser Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Ala Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro (2) INFORMATION FOR SEQ ID N0:63:
(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 N0:63:

Pro Met Thr Gln Thr Thr Ser Leu Lys Thr Ser Trp Val Asn Cys Ser Asn Met Ile Asp Glu Ile Ile Thr His Leu Lys Gln Pro Pro Leu Pro ' 20 25 30 Leu Leu Asp Phe Asn Asn Leu Asn Gly Glu Asp Gln Asp Ile Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu Glu Ala Phe Asn Arg Ala Val Lys Ser Leu Gln Asn Ala Ser Ala Ile Glu Ser Ile Leu Lys Asn Leu Leu Pro Cys Leu Pro Leu Ala Thr Ala Ala Pro Thr Arg His Pro Ile His Ile Lys Asp Gly Asp Trp Asn Gly Ile Phe Arg Arg Lys Leu Thr Phe Tyr Leu Lys Thr Leu Glu Asn Ala Gln Ala Gln Gln Thr Thr Leu Ser Leu Ala Ile Phe Tyr Val Glu Gly Gly Gly Gly Ser Pro Gly Glu Pro Ser Gly Pro Ile Ser Thr Ile Asn Pro Ser Pro Pro Ser Lys Glu Ser His Lys Ser Pro Asn Met Ala Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Ala Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro (2} INFORMATION FOR SEQ ID N0:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 407 base pairs (B} TYPE: nucleic acid (C} STRANDEDNESS: single (D) TOPOLOGY: linear (xi) SEQUENCE DESCRIPTION: SEQ ID N0:64:
CCATGGCTAA CTGCTCTATA ATGATCGATG AAATTATACA TCACTTAAAG AGACCACCTG
CACCTTTGCT GGACCCGAAC AACCTCAATG ACGAAGACGT CTCTATCCTG ATGGATCGAA

ACCTTCGACT TCCAAACCTG GAGAGCTTCG TAAGGGCTGT CAAGAACTTA GAAAATGCAT

CAGGTATTGA GGCAATTCTT CGTAATCTCC AACCATGTCT GCCCTCTGCC ACGGCCGCAC

CCTCTCGACA TCCAATCATC ATCAAGGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA

CGTTCTATCT GGTTACCCTT GAGCAAGCGC AGGAACAACA GTACGTAGAG GGCGGTGGAG

GCTCCCCGGG TGGTGGTTCT GGCGGCGGCT CCAACATGTA AGGTACC

(2) INFORMATION FOR SEQ ID N0:65:
(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 N0:65:
CCATGGCTAA CTGCTCTATA ATGATCGATG AAATTATACA TCACTTAAAG AGACCACCTG
CACCTTTGCT GGACCCGAAC AACCTCAATG ACGAAGACGT CTCTATCCTG ATGGATCGAA

ACCTTCGACT TCCAAACCTG GAGAGCTTCG TAAGGGCTGT CAAGAACTTA GAAAATGCAT

CAGGTATTGA GGCAATTCTT CGTAATCTCC AACCATGTCT GCCCTCTGCC ACGGCCGCAC

CCTCTCGACA TCCAATCATC ATCAAGGCAG GTGACTGGCA AGAATTCCGG GAAAAACTGA

CGTTCTATCT GGTTACCCTT GAGCAAGCGC AGGAACAACA GTACGTAGAG GGCGGTGGAG

GCTCCCCGGG TGAACCGTCT GGTCCAATCT CTACTATCAA CCCGTCTCCT CCGTCTAAAG

AATCTCATAA ATCTCCAAAC ATGTAAGGTA CC

Claims (39)

WHAT IS CLAIMED IS:

1. A chimeric protein comprising a flt3 agonist and a hematopoietic growth factor.

2. A chimeric protein comprising a polypeptide having a formula selected from the group consisting of:
R1-L-R2, R2-L-R1, R1-R2, R2-L-R1, Met-Ala-R1-L-R2, Met-Ala-R2-L-R1, Met-Ala-R1-R2, Met-Ala-R2-R1, Met-R1-L-R2, Met-R2-L-R1, Met-R1-R2, Met-R2-R1, Ala-R1-L-R2, Ala-R2-L-R1, Ala-R1-R2 and Ala-R2-R1;

wherein in R1 is a flt3 ligand;
R2 is a hematopoietic growth factor; and L is a linker capable of linking R1 to R2.

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, ~PO, 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 Ser17.

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.

38. 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.