Compositions and methods using unbound MPL receptor for stimulating platelet production.

Field pf the Invention

5

The present invention relates to the stimulation and growth of cells, particularly mega aryocytes and platelets, and specifically to the use of the so-called MPL receptor as an inducer of 0 megakaryocyte differentiation into platelets. The present invention also relates to compositions capable of bringing about such platelet production in vivo .

Background of the Invention 5

At least two broad areas of research are involved in the present invention. The first relates to the development and production of platelets from megakaryocytes and the second relates to a polypeptide 0 member of a growth factor receptor family, referred to herein as the MPL receptor. Each of these areas of research will now be outlined in the following.

Platelet Production from Meσakarvocvtes 5

Blood platelets are circulating cells that are crucial for the prevention of bleeding and for blood coagulation. Megakaryocytes are the cellular source of platelets and arise from a common bone marrow precursor 0 cell which gives rise to all hematopoietic cell lineages. This common precursor cell is known as the pluripotent stem cell or PPSC.

A hierarchy of megakaryocytic progenitor cells 5 has been defined based on the time of appearance and size of megakaryocyte (MK) colonies appearing in in

vitro culture systems in response to appropriate growth factors . The burst-forming unit megakaryocyte (BFU-MK) is the most primitive megakaryocyte progenitor cell. BFU-MK are thought to ultimately produce numerous colony forming unit megakaryocytes (CFU-MK) , which are more differentiated MK progenitor cells.

As the MK undergo subsequent differentiation, MK cells lose the ability to undergo mitosis but acquire an ability to endoreduplicate. Endoreduplication is the phenomenon in cells of nuclear division in the absence of cell division. Endoreduplication ultimately results in an MK which is polyploid. Further MK maturation results in acquisition of cytoplasmic organelles and membrane constituents that characterize platelets.

Platelets are produced from mature MK's by a poorly defined process that has been suggested to be a consequence of MK physical fragmentation, or other mechanisms. Observations of extensive membranous structures within megakaryocytes has led to a model of platelet formation in which a demarcation membrane system outlines nascent platelets within the cell body. Another model of platelet formation has developed from observations that megakaryocytes will form long cytoplasmic processes constricted at platelet-sized intervals from which platelets presumably break off due to blood flow pressures in the marrow and/or in the lung. These cytoplasmic processes were termed proplatelets by Becker and DeBruyn to reflect their presumed precursor role in platelet formation. See Becker and DeBruyn, Amer. J. Anat . 145: 183 (1976) .

FIG. 1 presents an overview of the various precursor cells involved in megakaryocyte and platelet development. The cell at the far left-hand side of the

figure may be considered a PPSC, and the additional cells to the right of the PPSC in the figure may be thought of as BFU-MK, followed by CFU-MK. The cell that is undergoing endomitosis, which is located immediately to the right of the PPSC in the figure, is a mature megakaryocyte cell. As a result of endomitosis, this cell has become polyploid. The next structure to the right includes the long cytoplasmic processes that are constricted at platelet-sized intervals emerging from the polyploid nucleus of the mature megakaryocyte cell. In the far right-hand side of the figure are shown a number of platelets that have been produced by fragmentation of the cytoplasmic processes in the immediately preceding megakaryocyte cell.

The following is a summary of some prior publications relating to the above description of megakaryocyte maturation and the production of platelets:

1. Williams, N. and Levine, R.F., British Journal of Haematology 52: 173-180 (1982) .

2. Levin, J., Molecular Biology and Differentiation of Megakaryocytes, pub. Wiley-Liss, Inc.: 1-10 (1990) .

3. Gewirtz, A.M., The Biology of Hematopoiesis, pub. Wiley-Liss, Inc.: 123-132 (1990) .

4. Han, Z.C., et al . , Int. J. Hematol. 54: 3-14 (1991) .

5. Nieuwenhuis, H.K. and Sixma, J., New Eng. J. of Med. 327: 1812-1813 (1992) .

Long, M., Stem Cells 11: 33-40 (1993)

B. Regulation of Platelet Formation

A large body of data generated in many laboratories indicates that platelet production is regulated by humoral factors . The complexity of this biological process was not originally appreciated and currently it appears that a number of human growth factors possess this capability.

Megakaryocyte regulation occurs at multiple cellular levels. A number of cytokine cell proliferation factors amplify platelet production by expanding the progenitor cell pool. A second group of humoral growth factors serves as maturation factors acting on more differentiated cells to promote endoreduplication. In addition, there appear to be two independent biofeedback loops regulating these processes.

Several lineage nonspecific hematopoietic growth factors exert an important effect on MK maturation. Granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-3 (IL-3), IL-6, IL-11, leukemia inhibitory factor (LIF) , and erythropoietin (EPO) each individually promote human MK maturation in vitro as determined by their effects on MK size, number, or ploidy. The MK maturational effects of LIF, IL-6, and IL-11 are either partially (LIF and IL-6) or totally (IL-11) additive to those of IL-3. Such data suggest that combinations of cytokines may be necessary to promote MK maturation in vivo .

The following is a summary of some prior publications relating to the regulation of megakaryocyte and platelet production:

7. Hoffman, R. et al., Blood Cells 13: 75-86 (1987) .

8. Murphy, M.J., Hematology/Oncology Clinics of North America 3 (3) : 465-478 (1988) .

9. Hoffman, R., Blood 74 (4) : 1196-1212 (1989) .

10. Mazur, E.M. and Cohen, J.L., Clin. Pharmacol. Ther., 46 (3) : 250-256 (1989) .

11. Gewirtz, A.M. and Calabretta, B., Int. J. Cell Cloning 8: 267-276 (1990) .

12. Williams, N., Progress in Growth Factor Research 2: 81-95 (1990) .

13. Gordon, M.S. and Hoffman, R., Blood 80 (2) : 302-307

(1992) .

14. Hunt, P. et al. , Exp. Hematol. 21: 372-281 (1993) .

15. Hunt, P. et al., Exp. Hematol. 21: 1295-1304

(1993) .

C. The MPL Receptor

The myeloproliferative leukemia virus (MPLV) is a murine replication-defective retrovirus that causes acute leukemia in infected mammals . It has been discovered that a gene expressed by MPLV consists of a part of the gene that encodes the retroviral envelope (or external protein coat) of the virus fused to a sequence that is related to the cytokine receptor family, including the receptors for GM-CSF, G-CSF, and EPO.

Expression of the MPLV gene described above has the interesting biological property of causing murine progenitor cells of various types to immediately acquire growth factor independence for both proliferation and terminal maturation. Moreover, some cultures of bone marrow cells acutely transformed by MPLV contained megakaryocytes, suggesting a connection between the MPLV gene and megakaryocyte growth and differentiation.

It is now recognized that the MPLV viral gene (referred to as v-MPL) has a homolog in mammalian cells, which is referred to as a cellular MPL gene (or c-MPL) . Using v-MPL-derived probes, a cDNA corresponding to the human c-MPL gene was cloned. See PCT published application WO 92/07074 (published April 30, 1992; discussed below) . Sequence analysis has shown that the protein encoded by the c-MPL gene product belongs to the highly conserved cytokine receptor superfamily, just like the homologous v-MPL gene product.

This cellular gene, c-MPL, is thought to play a functional role in hematopoiesis based on the observation that its expression was found in bone marrow, spleen, and fetal liver from normal mice by Northern blot analysis, but not in other tissues. In particular, c-MPL is expressed on megakaryocytes. It has also been shown that the human cellular gene, human c-MPL, is expressed in purified megakaryocytes, platelets and other cells that express the CD34 antigen, which is indicative of early hematopoietic progenitor cells. Furthermore, exposure of CD34 positive cells to synthetic oligodeoxynucleotides that are anti-sense to the c-MPL mRNA or message significantly inhibits the colony forming ability of CFU-MK megakaryocyte

progenitors, but has no effect on erythroid or granulomacrophage progenitors .

In total, the above data and observations suggest that the c-MPL-encoded protein could be the receptor for a cytokine specific for regulating megakaryocytopoiesis . In other words, c-MPL appears to encode a cell surface receptor, referred to herein as the MPL receptor, that binds to a ligand, which activates the receptor, possibly leading to production of megakaryocytes.

PCT patent publication WO 92/07074 is directed to the sequence of the protein produced by the c-MPL gene, from both human and murine sources. This gene product, which is thought to be a receptor as explained above, is made up of at least three general regions or domains: an extracellular domain, a transmembrane domain, and an intracellular (or cytoplasmic) domain. Attached together, these domains make up the intact MPL receptor. This PCT publication also refers to a soluble form of the receptor that substantially corresponds to the extracellular domain of the mature c-MPL protein. The intracellular domain contains a hydrophobic region that, when attached via the transmembrane region to the extracellular domain of the protein, renders the overall protein subject to aggregation and insolubility. On the other hand, when the extracellular domain of the c-MPL gene product is separated from the transmembrane domain and the intracellular domain, it becomes soluble, hence the extracellular form of the protein is referred to as a "soluble" form of the receptor.

A number of researchers are currently pursuing the isolation and characterization of the putative ligand which binds specifically to the c-MPL receptor.

It is expected that such ligand will stimulate the production of mature megakaryocytes and/or platelets. To date, however, no one has reported the purification or final structure of such a ligand. On the other hand, according to the prior publications relating to the MPL receptor (i.e., the intact c-MPL gene product), the soluble form of the MPL receptor would be expected to bind to circulating free MPL ligand, thus inhibiting the formation of megakaryocytes and/or platelets.

The following is a summary of some prior publications relating to the above description of the v-MPL and c-MPL receptors and genes:

16. Wendling, F., et al. , Leukemia 3 (7) : 475-480 (1989) .

17. Wendling, F., et al., Blood 73 (5) : 1161-1167 (1989) .

18. Souyri, M., et al., Cell 63: 1137-1147 (1990)

19. Vigon, I., et al., Proc. Natl. Acad. Sci. USA 89: 5640-5644 (1992) .

20. Skoda, R.C., et al. , The EMBO Journal 12 (7) : 2645-2653 (1993) .

21. Ogawa, M. Blood 81 (11) : 2844-2853 (1993) .

22. Methia, N., et al., Blood 82 (5) : 1395-1401 (1993)

23. Wendling, F, et al. , Blood 80: 246a (1993) .

D. The need for an agent capable of stimulating latelet production-

It has been reported recently that platelet transfusions are being administered at an ever increasing rate at medical centers in North America, Western Europe, and Japan. See Gordon, M.S. and Hoffman, R., Blood 80 (2) : 302-307 (1992) . This increase appears to be due in large measure to advances in medical technology and greater access to such technologies as cardiac surgery and bone marrow, heart, and liver transplantation. Dose intensification as a means of delivering therapies to cancer patients and the HIV-1 epidemic have also contributed to the heavy demand on the platelet supply.

Platelet usage carries with it the possibility of transmission of the many blood-born infectious diseases as well as alloimmunization. Moreover, the production of purified platelets is an expensive endeavor and hence the increasing use of such platelets increases overall medical costs. As a result, there exists an acute need for new and improved methods for producing platelets for human uses.

Exemplary prior approaches to enhancing platelet production are described in the following:

U.S. patent 5,032,396 reports that interleukin-7 (IL-7) is capable of stimulating platelet production. Interleukin-7 is also known as lymphopoietin-1 and is a lymphopoietic growth factor capable of stimulating growth of B- and T-cell progenitors in bone marrow. Published PCT application serial number 88/03747, filed October 19, 1988 and

European patent application number 88309977.2, filed

October 24, 1988 disclose DNA's, vectors, and related processes for producing mammalian IL-7 proteins by recombinant DNA technology. The data presented in the U.S. patent show that IL-7 can increase circulating platelets in normal and sublethally irradiated mice.

U.S. patent 5,087,448 discloses that megakaryocytes and platelets can be stimulated to proliferate in mammals by treating them with interleukin-6. Recombinant human interleukin-6 is a 26,000 molecular weight glycoprotein with multiple biological activities. The data presented in this patent show that IL-6 has an effect of increasing colonies of megakaryocytes in vitro .

Neither of the above-cited patents mentions anything with respect to the MPL receptor, which is involved in the present invention.

In spite of the above disclosures, there remains a strong need for new stimulators of megakaryocytes and/or platelets in mammals.

sυπimgiry pf the Invention

It is an object of the present invention to provide a means of stimulating production of megakaryocytes and/or platelets in vivo in a mammal in need thereof.

It is another object of the present invention to provide a treatment for platelet deficiencies in mammals, such as a human in need of such treatment.

- li ¬ lt is yet another object of the present invention to provide compositions for treating platelet deficiencies in mammals, such as humans.

These and other objects of the present invention as will hereinafter be described in greater detail, have been achieved by the discovery by the present inventors that administration of an unbound MPL receptor (defined below) to a mammal, results in an increased number of platelets in such mammal. The present invention also provides compositions for inducing platelet production, comprising an effective quantity of an unbound MPL receptor in admixture with one or more pharmaceutically acceptable diluents, carriers or excipients, as well as methods of using an unbound MPL receptor to prepare pharmaceutical compositions for inducing and enhancing platelet production in mammals.

Brief Description of the Figures

Numerons features and advantages of the present invention will become apparent upon review of the figures, wherein:

FIG. 1 depicts an overview of development and maturation of megakaryocytes and platelets.

FIG. 2 shows a schematic comparison of the domains of MPL-P (human) and Mpl (murine) . Additionally, a schematic depiction of an exemplary soluble form of the receptor is shown (Mpl-S) .

FIG. 3 shows data demonstrating that the addition of soluble MPL receptor (MPL-X) to

megakaryocyte cultures enhances the development of proplatelet formations .

FIG. 4 presents data showing the effect of MPL-X in vivo on platelet counts in a mouse model.

FIG. 5 presents data showing the effect of MPL-X in vivo on white blood cell (WBC) counts in a mouse model.

FIG. 6 presents data showing the effect of MPL-X in vivo on red blood cell (RBC) counts in a mouse model.

Detailed Description of the Invention

Additional aspects and advantages of the invention will be apparent to those skilled in the art upon consideration of the following detailed description, which details the practice of the invention.

The present invention is based on the unexpected discovery that MPL receptor, in unbound form, is capable of stimulating an increase in the number of platelets in vivo in mammals. This is believed to be due to an increased rate of megakaryocyte fragmentation into platelets. While it was previously appreciated that the MPL receptor might have some involvement in egakaryo-cytopoiesis, the understanding in the art was that when the receptor was bound to a megakaryocyte cell, it required stimulation by a ligand to cause the megakaryocyte to mature and/or to produce platelets. Based on the above hypothesis, it was a logical expectation that MPL receptor not attached to a cell

surface (i.e., "unbound MPL receptor") would compete for ligand with the MPL receptor bound to the cell, thereby removing ligand that would normally stimulate the cell to produce megakaryocytes and platelets, with the final effect being fewer cells proceeding to produce megakaryocytes and platelets.

The present inventors, however, have discovered that the opposite of the above-described expected result is actually observed. That is, in in vivo experiments in mammals, unbound MPL receptor has the effect of enhancing the production of megakaryocytes and platelets .

While not wishing to be bound by any particular mechanism or theory of action, the present inventors have formulated a hypothesis which may explain the effects described herein. This hypothesis is that the ligand that normally stimulates the bound MPL receptor produces mature megakaryocytes from less mature cells (e.g., PPSC, BFU-MK, CFU-MK) , but that once these mature megakaryocytes are produced, in order to proceed to form proplatelets, the ligand must be removed. If the ligand is not removed from the mature megakaryocyte, this megakaryocyte remains in the mature form rather than producing platelets.

Using the above hypothesis as a model, the administration of unbound MPL receptor would serve to allow mature megakaryocytes to proceed to form proplatelets and then fragment into platelets . At the same time, removal of the MPL ligand by the unbound MPL receptor may also inhibit the formation of additional mature megakaryocytes . If this hypothetical model is correct, it leads to the conclusion that even greater platelet formation might be achieved upon administration

of both the ligand and the unbound MPL receptor, since such administration would result in both enhanced production of mature megakaryocytes as well as stimulation of the production of platelets from the mature megakaryocytes. Sequential administration of ligand followed by unbound receptor might even further enhance the effect. In any case, the present inventors believe that this discovery is completely unexpected in view of the prior models of platelet formation and MPL receptor/ligand activity.

Definitions

The following definitions explain the meaning of key terms and phrases in the claims herein.

By "increasing the number of platelets" is meant that the number of platelets is significantly elevated above the normal range of platelets in the particular mammal involved. The elevation of platelet counts may occur in a time-dependent manner, and may be cyclical, increasing and then constant or decreasing, or constant, etc.

For example, with reference to FIG. 4, it appears that there are at least three cycles of elevated platelet count, with the first occurring at about seven to eight days, the second and more enhanced cycle occurring at between nineteen and twenty days, and the third cycle occurring at about twenty-eight to twenty- nine days . These cycles occurred in several mice in a particular model system, and may or may not be duplicated in other mammalian systems or models.

Preferably, the elevation in platelet count will be at least about forty to fifty percent above the average of the normal range, but may be considerably higher, such as up to about a two-fold increase as compared to the normal average number of platelets. It should be born in mind that any significant increase, such as, for example, five to ten percent, might be clinically sufficient in a given situation. The increase in platelet count is a function of, among other variables, the dose of the unbound MPL receptor administered to the mammal, and so can be altered over a wide range.

Platelets may be counted by any of various standard methods. One exemplary method involves the use of a commercially available blood cell counter. Such instruments "count" particles and classify them into cell types based essentially on size. Instruments such as the Sysmex or the Coulter Counter are exemplary. For a discussion of such methods, see Bloom, A.L. and

Thomas, D.P. (eds.), Haemostasis and Thrombosis (Second Edition) Churchill Livingstone: 936 (1987) .

The "mammal" to be treated is not specifically limited, but is preferably a human. Other mammals that might be treated include dogs, cats, cows, horses, etc.. Cross-species activity (i.e., activity of an MPL receptor from one species in another species) is also expected, especially if there is a high (e.g., equal to or above around 80%) homology between the MPL receptors from the two species, and is therefor another aspect of the present invention. For example, murine unbound MPL receptor could be used to increase the number of platelets in a human. However, it is expected that the greatest activity will be achieved using the same species' MPL receptor as the species being treated.

"Administration" may be carried out by any convenient method. For example, bolus injection, continuous infusion, sustained release from implants, or any other suitable technique could be used. Intravenous administration is preferred. Multiple administrations of unbound MPL receptor are also contemplated.

A "platelet number increasing effective amount" is an amount sufficient to significantly raise the number of platelets in a particular mammal. If necessary or desired, this amount can be appropriately determined in preliminary trials, without undue experimentation, and depending upon the level of increase desired in the mammal being treated. Generally, doses of 1 μg to 100 μg per kilogram per day, preferably 10 μg to 100 μg per kilogram per day for one to twenty days can be expected to induce a therapeutically significant biological effect. The unbound MPL receptor may be administered each day for a period of days, all at once on a first day followed by one or more days without administration, etc. Preferably, bolus injections of 10 μg per kilogram per day can be given at 3 - day intervals as a stimulator of platelet production.

In addition to the unbound MPL receptor, one or more additional lymphokines or cytokines may be administered simultaneously or sequentially with the MPL receptors of the present invention. Such lymphokines and/or cytokines may be, for example, IL-1 alpha, IL-1 beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-11, colony stimulating factor-1 (CSF-1), GM-CSF, granulocyte colony stimulating factor (G-CSF), EPO, interferon-alpha (IFN- alpha) , IFN-beta, or IFN-gamma. It is also possible to include the MPL ligand ,once it is available. Any of

these may work additively or synergistically with the unbound MPL receptors .

By "unbound MPL receptor" is meant an MPL receptor molecule that is free from the ordinary cellular constituents, especially the cell membrane. Preferably, the unbound receptor will be the so-called "soluble" form of the receptor. The "soluble" form of the MPL receptor may be any such form known to or subsequently developed by those skilled in the art.

Generally, the soluble form of the receptor will be one which substantially lacks the transmembrane domain of the receptor molecule.

It is known that the transmembrane domain of the MPL receptor is very hydrophobic, and therefore it is highly preferred that substantially all of this domain of the MPL receptor be removed for purposes of this invention. Although the intracellular domain of the MPL receptor is not considered insoluble, it is nevertheless preferred that this domain also be substantially removed from the MPL receptor for purposes of this invention.

In summary, by "soluble" is meant that the material is not bound to a cell, and preferably includes substantially none (i.e., fewer than about 10 residues) of the transmembrane or intracellular domains of the full-length molecule. The soluble MPL receptor (which is also referred to herein as MPL-X) is capable of being dissolved in aqueous solution as well as body fluids, such as blood, serum, saliva, cerebrospinal fluid, urine, etc. It is also possible to use the extracellular domain of the MPL receptor directly attached to the intracellular domain of the receptor.

As used herein, "MPL receptor" preferably refers to the

cellular form of this receptor (i.e., the c-MPL gene product) rather than the virally-derived form of the gene (i.e., v-MPL) .

The intact MPL receptor and its constituent domains are described in detail in published PCT publication WO92/07074, published April 30, 1992. Further details on the murine form of the MPL receptor are found in Vigon, et al. , EMBO 8: 2607 - 2615 (1993) . In addition, SEQ. ID NOS. 1 and 2 herein present the full length sequences of the murine MPL receptor gene and protein, respectively, while SEQ. ID NOS. 3 and 4 present the full length sequences of the murine MPL receptor gene and protein, respectively.

In the murine sequence (SEQ. ID NO. 2), the domains are as follows:

signal peptide aa 1 (Met) through aa 18 (Ser) extracellular aa 19 (Gin) through aa 483 (Trp) transmembrane aa 484 (lie) through aa 505 (Leu) cytoplasmic aa 506 (Lys) through aa 626 (Pro)

Referring again to the murine sequence (SEQ. ID NO. 2), some preferred soluble sequences are:

aa 19 (± 10 aa) through aa 483 (± 10 aa) , and

aa 19 (+ 10 aa) through aa 484 (+ 10 aa) fused to aa 506 (± 10 aa) through aa 626 (± 10 aa) .

It is known that the human sequence has at least two forms, MPL-P and MPL-K. The P form has the amino acid and gene sequences shown in SEQ. ID NOS. 3 and 4, respectively. In the human MPL-P sequence, the domains are as follows :

signal peptide aa 1 (Met) through aa 25 (Ser) extracellular aa 26 (Gin) through aa 491 (Trp) transmembrane aa 492 (lie) through aa 513 (Leu) cytoplasmic aa 514 (Arg) through aa 635 (Pro)

Referring to the human sequence (SEQ. ID NO. 4), some preferred soluble sequences are:

aa 19 (± 10 aa) through aa 483 (± 10 aa) , and

aa 19 (± 10 aa) through aa 484 (+ 10 aa) fused to aa 506 (± 10 aa) through aa 626 (± 10 aa) .

With reference to FIG. 2, one example of a soluble form of the receptor is depicted (Mpl-S) . It might be possible to remove even a larger portion of cytokine receptor domain II (CRD II) from either of the human or murine sequences and still have a soluble form of the receptor with platelet enhancing activity. Thus, the present inventors also contemplate the use of soluble receptors wherein substantially all of the CRD II domain is removed from the MPL receptor to enhance platelet production in a patient.

As exemplified above, based on the known MPL sequences and the prior publications in this area or routine experimentation, one of ordinary skill will be able to readily obtain a family of unbound murine or human MPL receptors for use in the context of this invention.

Various biologically active analogs of the foregoing proteins could also be employed in the methods and compositions of the present invention. As used herein, therefore, the term "MPL receptor" includes proteins having substantial amino acid sequence identity

to native mammalian MPL receptor and qualitatively equivalent biological activity, for example, in standard bioassays (e.g., assays of the receptor binding affinity or antibody binding affinity of the analog protein) . Particularly preferably, the analogs of the unbound MPL receptor of the present invention are based on the soluble form of the protein. The MPL receptor may further be in pegylated form or fused to other cytokine proteins or fragments thereof, e.g., IL-3, IL-6, IL-11, GM-CSF, EPO, and the like.

Preferred methods for producing mammalian MPL receptors of the present invention involve recombinant expression in mammalian cells, although such proteins can also be produced recombinantly using insect cells, yeast, bacteria, or other cells under the control of appropriate promoters. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described by Powels et al., Cloning Vectors: A Laboratory Manual (Elsevier New York, 1985) . Various mammalian cell culture systems can be employed to express recombinant protein.

Examples of mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23: 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, 293, HeLa and BHK cell lines. Mammalian expression vectors may comprise nontranscribed elements such as an origin of replication, a suitable promoter and enhancer, and other 5" or 3' flanking nontranscribed sequences, and 5' or 3' nontranslated sequences, such as necessary ribosome binding sites; a polyadenylation site; splice donor and acceptor sequences; and termination sequences. DNA

sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites, may be used to provide the other genetic elements required for expression of a heterologous DNA sequence. Additional details regarding the use of mammalian high expression vectors to produce a recombinant mammalian MPL receptor are provided below. Exemplary vectors can be constructed as disclosed by Okayama and Berg, Mol. Cell. Biol . 3:280 (1983); Cosman et al., Nature 312:768 (1984); Cosman et al. , Mol.

Immunol. 23:935 (1986); and Clark et al . , U.S. Pat. No. 4,675,285.

The conditions to be treated by the methods and compositions of the present invention are generally those which involve an existing platelet deficiency or an expected platelet deficiency in the future (e.g., because of planned surgery) . The generic term for platelet deficiency is thrombocytopenia, and hence the methods and compositions of the present invention are generally available for treating thrombocytopenias .

Thrombocytopenias (platelet deficiencies) may be present for various reasons, including chemotherapy, radiation therapy, surgery, accidental blood loss, and other specific disease conditions. Exemplary specific disease conditions that involve thrombocytopenia and may be treated in accordance with this invention are: aplastic anemia, idiopathic thrombocytopenia, and certain metastatic tumors which result in thrombo¬ cytopenia. Also, certain treatments for AIDS result in thrombocytopenia (e.g., AZT) . Certain wound healing disorders might also benefit from an increase in platelet numbers .

With regard to anticipated platelet deficiencies, e.g., due to future surgery, the MPL receptor could be administered several days to several hours prior to the need for platelets. With regard to acute situations, e.g., accidental and massive blood loss, the MPL receptor could be administered along with blood or purified platelets .

The specific dosage amount of unbound MPL receptor to be administered in connection with these conditions will be affected by a number of variables, including: the weight, sex, age, and general condition of the patient to be treated; the nature of the condition to be treated; the mode of administration; the urgency of the situation; and others. In acute situations, the dose to be administered will generally be larger than with chronic conditions or conditions with lesser platelet deficiency.

The compositions of the present invention include a "pharmaceutically acceptable carrier" in which the unbound receptor is soluble. The carrier material may be water for injection, preferably supplemented with other materials common in solutions for administration to mammals. Typically, an MPL receptor therapeutic will be administered in the form of a composition comprising purified protein in conjunction with physiologically acceptable carriers, excipients, or diluents. Neutral buffered saline or saline mixed with serum albumin are exemplary appropriate diluents. Preferably, the product is formulated as a lyophilizate using appropriate excipient solutions (e.g., sucrose) as diluents. Other standard carriers, diluents, and excipients may be included as desired.

EXAMPLES

The following examples are included to more fully illustrate the present invention. Additionally, these examples provide preferred embodiments of the invention, but are not meant to limit the scope thereof, unless so indicated. Standard methods for many of the procedures described in the following examples, or suitable alternative procedures, are provided in widely recognized manuals of molecular biology such as, for example, Sambrook et al . , "Molecular Cloning," Second Edition, Cold Spring Harbor Laboratory Press (1987) and in Ausubel et al . , (Eds), "Current Protocols in Molecular Biology, " Greene associates/Wiley Interscience, New York (1990) .

EXAMPLE 1

Isolation of nucleic acid sequences encoding an MPL receptor

The isolation of clones encoding the murine (Vigon et al. Oncogene 8:2607-2615 (1993) and (Skoda et al., EMBO) 12 (7) : 2645-2653 (1993)) and human (Vigon et al., DNAS 89: 5640-5644 (1992)) isoforms of the MPL receptor have been reported. The DNA sequence of these are available from the GenBank database as accession numbers X73677 and 222657 for the murine receptor and M90102 and M90103 for the K and P isoforms of the human receptor. It has further been reported that spleen, bone marrow, placenta and fetal liver are all tissues which express mRNA for MPL. Cell lines which express MPL include HEL. It thus becomes clear to one skilled in the art, how to isolate clones encoding MPL. cDNA could be generated from one of the above tissue sources or cell line, or from another source identified to

express MPL by Northern blot analysis of RNA or Western blot analysis of protein.

This cDNA serves as a source of material for the generation of a cDNA library in an appropriate vector. This library is screened by hybridization to a nucleic acid probe to identify cells containing the MPL gene. Alternatively the cDNA library is screened for expression of MPL using an antibody. This antibody is generated against a synthetic oligo-peptide corres¬ ponding to the MPL sequence.

The cDNA can also serve as a template for PCR amplification of the MPL gene. The primers are chosen from sequences in the MPL gene and may incorporate additional sequences to aid in the cloning and expression of MPL.

EXAMPLE 2

Isolation of clones for expression of soluble murine MPL

Using a clone containing MPL as identified above, or cDNA from a source capable of expressing MPL, the PCR technique is used to obtain a clone for expression of soluble MPL. Primers for PCR amplification of murine MPL may be of the form:

5' primer: TAC AAG CTT GCC GTC ATC ATG CCC TCT TGG GCC CTC (SEQ. ID NO. 5) ; and

3' primer:

ACT TCT AGA CTA TCA AGC AGT CTC GGA GCT GGA (SEQ. ID NO. 6)

PCR reactions are carried out using 1 μl of a cDNA reaction mix, 5 pmol of each of the above oligonucleotides, 10 mM Tris HC1 (pH 8.3), 50 mM KC1, 1.5 mM Mg CI ₂ , 200 μM each dNTP and 1 unit of Tag polymerase . Amplification is for 35 cycles of 30 sec. at 94°C, 30 sec. at 50°C, 1 min at 72°C. DNA is then purified by agarose gel electrophoresis, digested with Hind III and Xbal and ligated into the expression vector pDSR(x2 digested with Hind III and Xbal. Clones containing the desired insert are verified by DNA sequence analysis .

EXAMPLE 3

Expression of soluble murine MPL in CHO cells

The expression plasmid pDSR(X2-MPL-X contains sequences encoding murine MPL amino acids as shown in (SEQ. ID NOS. 7 and 8) . Ten micrograms of this plasmid were introduced into CHO cells by calcium phosphate mediated transfection (Wigler et al., Cell 11: 233 (1977) ) . Individual colonies were selected based upon expression of the dihydrofolate reductase gene from the vector. Expression of soluble MPL was monitored by RNA hybridization (Hunt et al. , Exp. Hematol, 19: 779 (1991)) and by Western blotting using an antibody generated to a synthetic peptide corresponding to amino acids 459 (Gly) to 475 (Val) of SEQ. ID NO. 2. Cell line B.l-18 was positive in these assays and was selected for further expansion. The cell line was adapted to 30nM Methotrexate (Mtx) to stimulate amplification of MPL expression. Roller bottles were innoculated with 2 x 10 ⁷ cells in 200 ml DMEM: Ham's F12 (1:1) supplemented with non-essential amino acids (NEAA) , 30nM Mtx and 5% fetal bovine serum (FBS)

(reagents from GIBCO, Grand Island, NY) . Upon reaching confluence in 3-4 days, the media was replaced with 200 ml DMEM: Ham's F12, NEAA, 30 nM Mtx with no serum. Conditioned media was harvested after 6-7 days and replaced with fresh serum-free media. Second and third harvests were collected.

EXAMPLE 4

Purification of soluble murine MPL

The recombinant, extracellular domain of the murine MPL gene product (m-MPL-X) was purified from CHO cell conditioned media by sequential ion exchange and hydroxyapatite chromatography. Conditioned media containing m-MPL-X was concentrated 10X by ultrafiltration with a 30,000 MW cutoff membrane (S10Y30, Amicon, Danvers, MA) and diafiltered against 10 mM Tris-HCL, pH 8.5. The concentrate was loaded onto a column of Q-Sepharose, fast flow (Pharmacia, Piscataway, NJ) , washed with 10 mM Tris-HCL, pH 8.5, and eluted with a linear gradient of 0 M-1.0 M NaCl in the same buffer. Fractions from the column were analyzed for m-MPL-X by SDS-PAGE and Western blotting, using an antiserum generated against purified m-MPL-X. Fractions containing m-MPL-X were pooled, diafiltered against 10 mM sodium phosphate, 0.01 mM CaCl2, pH 6.8, and applied to a hydroxyapatite column (HA-Ultragel, Sepracor, Malborough, MA) . The unbound fraction, containing purified m-MPL-X was diluted into PBS, sterile filtered, and aseptically dispensed in appropriate volumes at a final concentration of 0.10 mg/ml . The purified m-MPL-X was stored frozen at -80° C until use.

The Limulus Amebocyte Lysate assay (Associates of Cape Cod, Inc., Woods Hole, MA) was performed, and the final product was shown to be free of pyrogen (< 0.004 EU/mg) . Analysis of 20 μg of purified m-MPL-X by SDS-PAGE with Coomassie staining demonstrated the presence of a single band with apparent molecular weight of 64 KD. No other protein bands were detected.

EXAMPLE 5

The effect of MPL-X on proplatelet formation

Guinea pig megakaryocytes were purified and cultured at 5000 cells per well (96- well microtiter plate) with or without MPL-X (30 μg/ml; prepared as described above) for 18 hours. The basal media was Iscove's media supplemented either with BSA (100 μg/ml) or with 10% normal human heparinized plasma (pooled from AB donors) . After culture, cells were examined microscopically and scored for proplatelet formations as described by Hunt et al. See Hunt, P. et al., Exp. Hematol. 21: 372-281 (1993) and Hunt, P. et al., Exp. Hematol. 21: 1295-1304 (1993) . Data is presented as the mean of duplicate determinations +/- standard error of the mean (SEM) .

Guinea pig megakaryocytes cultured in serum- free media or in heparinized plasma have previously been shown to develop proplatelet formations after 18 hours of culture. See Hunt, P. et al. , Exp. Hematol. 21: 372- 281 (1993) and Hunt, P. et al., Exp. Hematol. 21: 1295- 1304 (1993) . As shown in FIG. 3, the addition of MPL-X to megakaryocyte cultures resulted in a ^' significant increase in the number of cells developing proplatelet

formations. This was observed whether the cells were cultured under serum-free conditions or in the presence of heparinized plasma.

EXAMPLE 6

The effect of MPL-X on in vi vo proplatelet formation

MPL-X was diluted to 100 μg/ml or 10 μg/ml into carrier (PBS + 0.2% normal Balb/c mouse sera) . In addition, 100 μg/ml of bovine serum albumin (BSA) or heat-inactivated MPL-X (98°C; 15 minutes) were used. Balb/c mice (female, 6-8 weeks of age, Charles River) were injected subcutaneously twice daily, with an 8 hour interval, with 0.5 ml of each test solution for up to 42 days. On the indicated days, animals were bled from the lateral tail vein through a small incision made with a scalpel blade. Twenty microliters of blood were collected and diluted immediately into manufacturer's diluent for the Sysmex cell analyzer (TOA Medical Electronics, Kobe, Japan) . White blood cell (WBC) , red blood cell (RBC) , -and platelet counts were obtained. Data are expressed as the mean of the indicated number of samples +/- SEM.

In order to determine directly the effect of MPL-X on platelet levels, the protein was injected twice daily into normal Balb/c mice. The data from two separate experiments were combined for presentation. ^' In Experiment 1, five mice per group were injected either with carrier, 10 μg/day MPL-X, or 100 μg/day MPL-X for 42 days. In Experiment 2, four mice per group were injected either with carrier, 100 μg/day MPL-X, 100 μg/day heat-inactivated MPL-X or 100 μg/day BSA for 29 days . Whenever data from both experiments were collected from the same time point, they were combined

for analysis (N=9, days 0, 4, 7, 11, 18; N=4 or 5, all other days) . As shown in FIG. 4, injection of 100 μg/day MPL-X resulted in a significant increase in platelet counts. The effect was first observed after 4-7 days of treatment where platelet levels reached 132% normal levels. Although by Day 11 platelet counts had returned to normal, with continued administration of MPL-X, they again rose reaching 219% of normal by Day 18. After 20 days of treatment, the platelet counts had dropped to 150% of normal. When the study was terminated at 42 days, platelet counts were still 130% of normal. As also shown in FIG. 4, administration of 10 μg/day of MPL-X, 100 μg/day of BSA or heat inactivated MPL-X had no effect on platelet counts . MPL-X administration had no effect on other blood cell parameters . Figures 5 and 6 illustrate the data on white blood cell counts and red blood cell counts.

These data indicate that the administration of MPL-x in vivo results in a significant increase in circulating platelets . The response appears to be cyclic with a periodicity of approximately 7-10 days.

No other blood cell parameter was affected at any time point measured.

While the present invention has been described above both generally and in terms of preferred embodiments, it is understood that variations and modifications will occur to those skilled in the art in light of the above description. Therefore, it is intended that the appended claims cover all such variations coming within the scope of the invention as claimed.

Additionally, the publications and other materials cited to illuminate the background of the invention, and in particular cases to provide additional details concerning its practice, are herein incorporated by reference.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANTS: Choi, Esther S. Ho o , Martha M. Hunt, Pamela Nichol, Janet L.

(ii) TITLE OF INVENTION: Compositions And Methods For

Stimulating Platelet Production

(iii) NUMBER OF SEQUENCES: 8

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Amgen Inc., U.S. Patent Operations/RRC

(B) STREET: 1840 DeHavilland Drive

(C) CITY: Thousand Oaks (D) STATE: CA

(F) ZIP: 91320-1789

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 805-447-4955

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

(A) LENGTH: 2046 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown .

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE: (A) NAME/KEY: CDS

(B) LOCATION: 8..1888

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

CCTCTTC ATG GTC ACC TCC TGC CTC CTC TTG GCC CTT CCA AAC CAG GCA 49 Met Val Thr Ser Cys Leu Leu Leu Ala Leu Pro Asn Gin Ala 1 5 10

CAA GTC ACC AGC CAA GAT GTC TTC TTG CTG GCC TTG GGC ACA GAG CCC 97

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

15 20 25 30

CTG AAC TGC TTC TCC CAA ACA TTT GAG GAC CTC ACC TGC TTC TGG GAT 145

Leu Asn Cys Phe Ser Gin Thr Phe Glu Asp Leu Thr Cys Phe Trp Asp

■ 35 40 45 GAG GAA GAG GCA GCA CCC AGT GGG ACA TAC CAG CTG CTG TAT GCC TAC 193

Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gin Leu Leu Tyr Ala Tyr

50 55 60

CGA GGA GAG AAG CCC CGT GCA TGC CCC CTG TAT TCC CAG AGT GTG CCC 2 1 Arg Gly Glu Lys Pro Arg Ala Cys Pro Leu Tyr Ser Gin Ser Val Pro 65 70 75

ACC TTT GGA ACC CGG TAT GTG TGC CAG TTT CCA GCC CAG GTA GAA GTG 289

Thr Phe Gly Thr Arg Tyr Val Cys Gin Phe Pro Ala Gin Val Glu Val 80 85 90

CGC CTC TTC TTT CCG CTG CAC CTC TGG GTG AAG AAT GTG TCC CTC AAC 337

Arg Leu Phe Phe Pro Leu His Leu Trp Val Lys Asn Val Ser Leu Asn

95 100 105 110

CAG ACT TTG ATC CAG CGG GTG CTG TTT GTG GAT AGT GTG GGC CTG CCA 385

Gin Thr Leu He Gin Arg Val Leu Phe Val Asp Ser Val Gly Leu Pro

115 120 125 GCT CCC CCC AGG GTC ATC AAG GCC AGG GGT GGG AGC CAA CCA GGG GAA 433

Ala Pro Pro Arg Val He Lys Ala Arg Gly Gly Ser Gin Pro Gly Glu

130 135 140

CTT CAG ATC CAC TGG GAG GCC CCT GCT CCT GAA ATC AGT GAC TTC CTG 481 Leu Gin He His Trp Glu Ala Pro Ala Pro Glu He Ser Asp Phe Leu 145 150 155

AGG CAT GAA CTC CGC TAT GGC CCC ACG GAT TCC AGC AAC GCC ACT GCC 529

Arg His Glu Leu Arg Tyr Gly Pro Thr Asp Ser Ser Asn Ala Thr Ala 160 165 170

CCC TCC GTC ATT CAG CTG CTC TCC ACA GAA ACC TGC TGC CCC ACT TTG 577

Pro Ser Val He Gin Leu Leu Ser Thr Glu Thr Cys Cys Pro Thr Leu

175 180 185 190

TGG ATG CCG AAC CCA GTC CCT GTT CTT GAC CAG CCT CCG TGT GTT CAT 625

Trp Met Pro Asn Pro Val Pro Val Leu Asp Gin Pro Pro Cys Val His

195 200 205 CCG ACA GCA TCC CAA CCG CAT GGA CCA GTG AGG ACC TCC CCA GCT GGA 673

Pro Thr Ala Ser Gin Pro His Gly Pro Val Arg Thr Ser Pro Ala Gly

210 215 220

GAA GCT CCA TTT CTG ACA GTG AAG GGT GGA AGC TGT CTC GTC TCA GGC 721 Glu Ala Pro Phe Leu Thr Val Lys Gly Gly Ser Cys Leu Val Ser Gly 225 230 235

CTC CAG GCT AGC AAA TCC TAC TGG CTC CAG CTA CGC AGC CAA CCC GAC 769 Leu Gin Ala Ser Lys Ser Tyr Trp Leu Gin Leu Arg Ser Gin Pro Asp 240 245 250 GGG GTC TCC CTT CGT GGC TCC TGG GGA CCC TGG TCC TTC CCT GTG ACT 817 Gly Val Ser Leu Arg Gly Ser Trp Gly Pro Trp Ser Phe Pro Val Thr 255 260 265 270

GTG GAT CTT CCA GGA GAT GCA GTG ACA ATT GGA CTT CAG TGC TTT ACC 865 Val Asp Leu Pro Gly Asp Ala Val Thr He Gly Leu Gin Cys Phe Thr

275 280 285

TTG GAT CTG AAG ATG GTC ACC TGC CAG TGG CAG CAA CAA GAC CGC ACT 913 Leu Asp Leu Lys Met Val Thr Cys Gin Trp Gin Gin Gin Asp Arg Thr 290 295 300

AGC TCC CAA GGC TTC TTC CGT CAC AGC AGG ACG AGG TGC TGC CCC ACA 961

Ser Ser Gin Gly Phe Phe Arg His Ser Arg Thr Arg Cys Cys Pro Thr

305 310 315

GAC AGG GAC CCC ACC TGG GAG AAA TGT GAA GAG GAG GAA CCG CGT CCA 1009

Asp Arg Asp Pro Thr Trp Glu Lys Cys Glu Glu Glu Glu Pro Arg Pro

320 325 330 GGA TCA CAG CCC GCT CTC GTC TCC CGC TGC CAC TTC AAG TCA CGA AAT 1057 Gly Ser Gin Pro Ala Leu Val Ser Arg Cys His Phe Lys Ser Arg Asn 335 340 345 350

GAC AGT GTT ATT CAC ATC CTT GTA GAG GTG ACC ACA GCG CAA GGT GCC 1105 Asp Ser Val He His He Leu Val Glu Val Thr Thr Ala Gin Gly Ala

355 360 365

GTT CAC AGC TAC CTG GGC TCC CCT TTT TGG ATC CAC CAG GCT GTG CTC 1153 Val His Ser Tyr Leu Gly Ser Pro Phe Trp He His Gin Ala Val Leu 370 375 380

CTT CCC ACC CCG AGC CTG CAC TGG AGG GAG GTC TCA AGT GGA AGG CTG 1201 Leu Pro Thr Pro Ser Leu His Trp Arg Glu Val Ser Ser Gly Arg Leu 385 390 395

GAG TTG GAG TGG CAG CAC CAG TCA TCT TGG GCA GCT CAA GAG ACC TGC 1249 Glu Leu Glu Trp Gin His Gin Ser Ser Trp Ala Ala Gin Glu Thr Cys 400 405 410 TAC CAG CTC CGG TAC ACG GGA GAA GGC CGT GAG GAC TGG AAG GTG CTG 1297 Tyr Gin Leu Arg Tyr Thr Gly Glu Gly Arg Glu Asp Trp Lys Val Leu 415 420 425 430

GAG CCA TCT CTC GGT GCC CGG GGA GGG ACC CTA GAG CTG CGC CCC CGA 1345 Glu Pro Ser Leu Gly Ala Arg Gly Gly Thr Leu Glu Leu Arg Pro Arg

435 440 445

GCT CGC TAC AGC TTG CAG CTG CGT GCC AGG CTC AAC GGC CCC ACC TAC 1393 Ala Arg Tyr Ser Leu Gin Leu Arg Ala Arg Leu Asn Gly Pro Thr Tyr 450 455 460

CAA GGT CCC TGG AGC GCC TGG TCT CCC CCA GCT AGG GTG TCC ACG GGC 1441 Gin Gly Pro Trp Ser Ala Trp Ser Pro Pro Ala Arg Val Ser Thr Gly 465 470 475 TCC GAG ACT GCT TGG ATC ACC TTG GTG ACT GCT CTG CTC CTG GTG CTG 1489 Ser Glu Thr Ala Trp He Thr Leu Val Thr Ala Leu Leu Leu Val Leu 480 485 490

AGC CTC AGT GCC CTT CTG GGC CTA CTG CTG CTA AAG TGG CAA TTT CCT 1537 Ser Leu Ser Ala Leu Leu Gly Leu Leu Leu Leu Lys Trp Gin Phe Pro 495 500 505 510

GCG CAC TAC AGG AGA CTG AGG CAT GCT TTG TGG CCC TCG CTT CCA GAC 1585 Ala His Tyr Arg Arg Leu Arg His Ala Leu Trp Pro Ser Leu Pro Asp 515 520 525

CTA CAC CGG GTC CTA GGC CAG TAC CTC AGA GAC ACT GCA GCC CTA AGT 1633

Leu His Arg Val Leu Gly Gin Tyr Leu Arg Asp Thr Ala Ala Leu Ser 530 535 540

CCT TCT AAG GCC ACG GTT ACC GAT AGC TGT GAA GAA GTG GAA CCC AGC 1681

Pro Ser Lys Ala Thr Val Thr Asp Ser Cys Glu Glu Val Glu Pro Ser 545 550 555 CTC CTG GAA ATC CTC CCT AAA TCC TCA GAG AGC ACT CCT TTA CCT CTG 1729 Leu Leu Glu He Leu Pro Lys Ser Ser Glu Ser Thr Pro Leu Pro Leu 560 565 570

TGT CCC TCC CAA CCT CAG ATG GAC TAC AGA GGA CTG CAA CCT TGC CTG 1777 Cys Pro Ser Gin Pro Gin Met Asp Tyr Arg Gly Leu Gin Pro Cys Leu 575 580 585 590

CGG ACC ATG CCC CTG TCT GTG TGT CCA CCC ATG GCT GAG ACG GGG TCC 1825 Arg Thr Met Pro Leu Ser Val Cys Pro Pro Met Ala Glu Thr Gly Ser 595 600 605

TGC TGC ACC ACA CAC ATT GCC AAC CAC TCC TAC CTA CCA CTA AGC TAT 1873 Cys Cys Thr Thr His He Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr 610 615 620

TGG CAG CAG CCC TGAAGGCAGT CCCCATGCTA CTGCAGACCT ATACATTCCT 1925 Trp Gin Gin Pro 625 ACACACTACC TTATCCATCC TCAACACCAT CCATTCTGTT GCCACCCCAC TCCCCCTCTG 1985

GCTTTATAAC ACTGATCACT CCAAGATGGC TGCTCACAAA TCCAGAGCTC TGTCTCTGCA 2045

G 2046

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 626 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Val Thr Ser Cys Leu Leu Leu Ala Leu Pro Asn Gin Ala Gin Val 1 5 10 15

Thr Ser Gin Asp Val Phe Leu Leu Ala Leu Gly Thr Glu Pro Leu Asn 20 25 30

Cys Phe Ser Gin Thr Phe Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu 35 40 45

Glu Ala Ala Pro Ser Gly Thr Tyr Gin Leu Leu Tyr Ala Tyr Arg Gly 50 55 60

Glu Lys Pro Arg Ala Cys Pro Leu Tyr Ser Gin Ser Val Pro Thr Phe 65 70 75 80 Gly Thr Arg Tyr Val Cys Gin Phe Pro Ala Gin Val Glu Val Arg Leu

85 90 95

Phe Phe Pro Leu His Leu Trp Val Lys Asn Val Ser Leu Asn Gin Thr 100 105 110

Leu He Gin Arg Val Leu Phe Val Asp Ser Val Gly Leu Pro Ala Pro

115 120 125

Pro Arg Val He Lys Ala Arg Gly Gly Ser Gin Pro Gly Glu Leu Gin 130 135 140

He His Trp Glu Ala Pro Ala Pro Glu He Ser Asp Phe Leu Arg His 145 _' • 150 155 160 Glu Leu Arg Tyr Gly Pro Thr Asp Ser Ser Asn Ala Thr Ala Pro Ser

165 170 175

Val He Gin Leu Leu Ser Thr Glu Thr Cys Cys Pro Thr Leu Trp Met 180 185 190

Pro Asn Pro Val Pro Val Leu Asp Gin Pro Pro Cys Val His Pro Thr 195 200 205

Ala Ser Gin Pro His Gly Pro Val Arg Thr Ser Pro Ala Gly Glu Ala 210 215 220

Pro Phe Leu Thr Val Lys Gly Gly Ser Cys Leu Val Ser Gly Leu Gin 225 230 235 240 Ala Ser Lys Ser Tyr Trp Leu Gin Leu Arg Ser Gin Pro Asp Gly Val

245 250 255

Ser Leu Arg Gly Ser Trp Gly Pro Trp Ser Phe Pro Val Thr Val Asp 260 265 270

Leu Pro Gly Asp Ala Val Thr He Gly Leu Gin Cys Phe Thr Leu Asp 275 280 285

Leu Lys Met Val Thr Cys Gin Trp Gin Gin Gin Asp Arg Thr Ser Ser 290 295 300

Gin Gly Phe Phe Arg His Ser Arg Thr Arg Cys Cys Pro Thr Asp Arg 305 310 315 320

Asp Pro Thr Trp Glu Lys Cys Glu Glu Glu Glu Pro Arg Pro Gly Ser 325 330 335 Gin Pro Ala Leu Val Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser

340 345 350

Val He His He Leu Val Glu Val Thr Thr Ala Gin Gly Ala Val His 355 360 365

Ser Tyr Leu Gly Ser Pro Phe Trp He His Gin Ala Val Leu Leu Pro 370 375 380

Thr Pro Ser Leu His Trp Arg Glu Val Ser Ser Gly Arg Leu Glu Leu 385 390 395 400

Glu Trp Gin His Gin Ser Ser Trp Ala Ala Gin Glu Thr Cys Tyr Gin 405 410 415 Leu Arg Tyr Thr Gly Glu Gly Arg Glu Asp Trp Lys Val Leu Glu Pro

420 425 430

Ser Leu Gly Ala Arg Gly Gly Thr Leu Glu Leu Arg Pro Arg Ala Arg 435 440 445

Tyr Ser Leu Gin Leu Arg Ala Arg Leu Asn Gly Pro Thr Tyr Gin Gly 450 455 460

Pro Trp Ser Ala Trp Ser Pro Pro Ala Arg Val Ser Thr Gly Ser Glu 465 470 475 480

Thr Ala Trp He Thr Leu Val Thr Ala Leu Leu Leu Val Leu Ser Leu 485 490 495 Ser Ala Leu Leu Gly Leu Leu Leu Leu Lys Trp Gin Phe Pro Ala His

500 505 510

Tyr Arg Arg Leu Arg His Ala Leu Trp Pro Ser Leu Pre Asp Leu His 515 520 525

Arg Val Leu Gly Gin Tyr Leu Arg Asp Thr Ala Ala Leu Ser Pro Ser 530 535 540

Lys Ala Thr Val Thr Asp Ser Cys Glu Glu Val Glu Pro Ser Leu Leu 545 550 555 560

Glu He Leu Pro Lys Ser Ser Glu Ser Thr Pro Leu Pro Leu Cys Pro 565 570 575 Ser Gin Pro Gin Met Asp Tyr Arg Gly Leu Gin Pro Cys Leu Arg Thr

580 585 590

Met Pro Leu Ser Val Cys Pro Pro Met Ala Glu Thr Gly Ser Cys Cys 595 600 605

Thr Thr His He Ala Asn His Ser Tyr Leu Pro Leu Ser Tyr Trp Gin 610 615 620

Gin Pro 625

(2) INFORMATION FOR SEQ ID NO:3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 1908 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 1..1908

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

ATG CCC TCC TGG GCC CTC TTC ATG GTC ACC TCC TGC CTC CTC CTG GCC 48 Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu Ala

1 5 10 15

CCT CAA AAC CTG GCC CAA GTC AGC AGC CAA GAT GTC TCC TTG CTG GCA 96

Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser Leu Leu Ala 20 25 30

TCA GAC TCA GAG CCC CTG AAG TGT TTC TCC CGA ACA TTT GAG GAC CTC 144

Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe Glu Asp Leu

35 40 45

ACT TGC TTC TGG GAT GAG GAA GAG GCA GCG CCC AGT GGG ACA TAC CAG 192

Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gin 50 55 60 CTG CTG TAT GCC TAC CCG CGG GAG AAG CCC CGT GCT TGC CCC CTG AGT 240

Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg Ala Cys Pro Leu Ser

65 70 75 80

TCC CAG AGC ATG CCC CAC TTT GGA ACC CGA TAC GTG TGC CAG TTT CCA 288 Ser Gin Ser Met Pro His Phe Gly Thr Arg Tyr Val Cys Gin Phe Pro

85 90 95

GAC CAG GAG GAA GTG CGT CTC TTC TTT CCG CTG CAC CTC TGG GTG AAG 336

Asp Gin Glu Glu Val Arg Leu Phe Phe Pro Leu His Leu Trp Val Lys 100 105 110

AAT GTG TTC CTA AAC CAG ACT CGG ACT CAG CGA GTC CTC TTT GTG GAC 384 Asn Val Phe Leu Asn Gin Thr Arg Thr Gin Arg Val Leu Phe Val Asp 115 120 125 AGT GTA GGC CTG CCG GCT CCC CCC AGT ATC ATC AAG GCC ATG GGT GGG 432 Ser Val Gly Leu Pro Ala Pro Pro Ser He He Lys Ala Met Gly Gly 130 135 140

AGC CAG CCA GGG GAA CTT CAG ATC AGC TGG GAG GAG CCA GCT CCA GAA 480 Ser Gin Pro Gly Glu Leu Gin He Ser Trp Glu Glu Pro Ala Pro Glu 145 150 155 160

ATC AGT GAT TTC CTG AGG TAC GAA CTC CGC TAT GGC CCC AGA GAT CCC 528 He Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro 165 170 175

AAG AAC TCC ACT GGT CCC ACG GTC ATA CAG CTG ATT GCC ACA GAA ACC 576

Lys Asn Ser Thr Gly Pro Thr Val He Gin Leu He Ala Thr Glu Thr

180 185 190

TGC TGC CCT GCT CTG CAG AGG CCT CAC TCA GCC TCT GCT CTG GAC CAG 624

Cys Cys Pro Ala Leu Gin Arg Pro His Ser Ala Ser Ala Leu Asp Gin

195 200 205 TCT CCA TGT GCT CAG CCC ACA ATG CCC TGG CAA GAT GGA CCA AAG CAG 672 Ser Pro Cys Ala Gin Pro Thr Met Pro Trp Gin Asp Gly Pro Lys Gin 210 215 220

ACC TCC CCA AGT AGA GAA GCT TCA GCT CTG ACA GCA GAG GGT GGA AGC 720 Thr Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu Gly Gly Ser 225 230 235 240

TGC CTC ATC TCA GGA CTC CAG CCT GGC AAC TCC TAC TGG CTG CAG CTG 768 Cys Leu He Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp Leu Gin Leu 245 250 255

CGC AGC GAA CCT GAT GGG ATC TCC CTC GGT GGC TCC TGG GGA TCC TGG 816

Arg Ser Glu Pro Asp Gly He Ser Leu Gly Gly Ser Trp Gly Ser Trp

260 265 270

TCC CTC CCT GTG ACT GTG GAC CTG CCT GGA GAT GCA GTG GCA CTT GGA 864

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

275 280 285 CTG CAA TGC TTT ACC TTG GAC CTG AAG AAT GTT ACC TGT CAA TGG CAG 912 Leu Gin Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys Gin Trp Gin 290 295 300

CAA CAG GAC CAT GCT AGC TCC CAA GGC TTC TTC TAC CAC AGC AGG GCA 960 Gin Gin Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His Ser Arg Ala 305 310 315 320

CGG TGC TGC CCC AGA GAC AGG TAC CCC ATC TGG GAG AAC TGC GAA GAG 1008 Arg Cys Cys Pro Arg Asp Arg Tyr Pro He Trp Glu Asn Cys Glu Glu 325 330 335

GAA GAG AAA ACA AAT CCA GGA CTA CAG ACC CCA CAG TTC TCT CGC TGC 1056

Glu Glu Lys Thr Asn Pro Gly Leu Gin Thr Pro Gin Phe Ser Arg Cys

340 345 350 CAC TTC AAG TCA CGA AAT GAC AGC ATT ATT CAC ATC CTT GTG GAG GTG 110

His Phe Lys Ser Arg Asn Asp Ser He He His He Leu Val Glu Val

355 360 365

ACC ACA GCC CCG GGT ACT GTT CAC AGC TAC CTG GGC TCC CCT TTC TGG 1152 Thr Thr Ala Pro Gly Thr Val His Ser Tyr Leu Gly Ser Pro Phe Trp 370 375 380

ATC CAC CAG GCT GTG CGC CTC CCC ACC CCA AAC TTG CAC TGG AGG GAG 1200

He His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His Trp Arg Glu 385 390 395 400

ATC TCC AGT GGG CAT CTG GAA TTG GAG TGG CAG CAC CCA TCG TCC TGG 1248

He Ser Ser Gly His Leu Glu Leu Glu Trp Gin His Pro Ser Ser Trp

405 410 415

GCA GCC CAA GAG ACC TGT TAT CAA CTC CGA TAC ACA GGA GAA GGC CAT 1296

Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly Glu Gly His

420 425 430 CAG GAC TGG AAG GTG CTG GAG CCG CCT CTC GGG GCC CGA GGA GGG ACC 1344

Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr

435 440 445

CTG GAG CTG CGC CCG CGA TCT CGC TAC CGT TTA CAG CTG CGC GCC AGG 1392 Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu Arg Ala Arg 450 455 460

CTC AAC GGC CCC ACC TAC CAA GGT CCC TGG AGC TCG TGG TCG GAC CCA 1440

Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp Ser Asp Pro 465 470 475 480

ACT AGG GTG GAG ACC GCC ACC GAG ACC GCC TGG ATC TCC TTG GTG ACC 1488

Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp He Ser Leu Val Thr

485 490 495

GCT CTG CAT CTA GTG CTG GGC CTC AGC GCC GTC CTG GGC CTG CTG CTG 1536

Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly Leu Leu Leu

500 505 510 CTG AGG TGG CAG TTT CCT GCA CAC TAC AGG AGA CTG AGG CAT GCC CTG 1584

Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu

515 520 525

TGG CCC TCA CTT CCA GAC CTG CAC CGG GTC CTA GGC CAG TAC CTT AGG 1632 Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin Tyr Leu Arg 530 535 540

GAC ACT GCA GCC CTG AGC CCG CCC AAG GCC ACA GTC TCA GAT ACC TGT 1680

Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser Asp Thr Cys 545 550 555 560

GAA GAA GTG GAA CCC AGC CTC CTT GAA ATC CTC CCC AAG TCC TCA GAG 1728 Glu Glu Val Glu Pro Ser Leu Leu Glu He Leu Pro Lys Ser Ser Glu 565 570 575 AGG ACT CCT TTG CCC CTG TGT TCC TCC CAG GCC CAG ATG GAC TAC CGA 1776 Arg Thr Pro Leu Pro Leu Cys Ser Ser Gin Ala Gin Met Asp Tyr Arg 580 585 590

AGA TTG CAG CCT TCT TGC CTG GGG ACC ATG CCC CTG TCT GTG TGC CCA 1824 Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser Val Cys Pro 595 600 605

CCC ATG GCT GAG TCA GGG TCC TGC TGT ACC ACC CAC ATT GCC AAC CAT 1872 Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His He Ala Asn His 610 615 620

TCC TAC CTA CCA CTA AGC TAT TGG CAG CAG CCT TG 1908

Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro 625 630 635

(2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 635 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

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

Met Pro Ser Trp Ala Leu Phe Met Val Thr Ser Cys Leu Leu Leu Ala

1 5 10 15

Pro Gin Asn Leu Ala Gin Val Ser Ser Gin Asp Val Ser Leu Leu Ala 20 ^* 25 30

Ser Asp Ser Glu Pro Leu Lys Cys Phe Ser Arg Thr Phe Glu Asp Leu 35 40 45

Thr Cys Phe Trp Asp Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gin

50 55 60 Leu Leu Tyr Ala Tyr Pro Arg Glu Lys Pro Arg Ala Cys Pro Leu Ser

65 70 75 80

Ser Gin Ser Met Pro His Phe Gly Thr Arg Tyr Val Cys Gin Phe Pro 85 90 95

Asp Gin Glu Glu Val Arg Leu Phe Phe Pro Leu His Leu Trp Val Lys 100 105 110

Asn Val Phe Leu Asn Gin Thr Arg Thr Gin Arg Val Leu Phe Val Asp 115 120 125

Ser Val Gly Leu Pro Ala Pro Pro Ser He He Lys Ala Met Gly Gly

130 135 140

Ser Gin Pro Gly Glu Leu Gin He Ser Trp Glu Glu Pro Ala Pro Glu 145 150 155 160

He Ser Asp Phe Leu Arg Tyr Glu Leu Arg Tyr Gly Pro Arg Asp Pro 165 170 175

Lys Asn Ser Thr Gly Pro Thr Val He Gin Leu He Ala Thr Glu Thr 180 185 190 Cys Cys Pro Ala Leu Gin Arg Pro His Ser Ala Ser Ala Leu Asp Gin 195 200 205

Ser Pro Cys Ala Gin Pro Thr Met Pro Trp Gin Asp Gly Pro Lys Gin 210 215 220

Thr Ser Pro Ser Arg Glu Ala Ser Ala Leu Thr Ala Glu Gly Gly Ser 225 230 235 240

Cys Leu He Ser Gly Leu Gin Pro Gly Asn Ser Tyr Trp Leu Gin Leu 245 250 255

Arg Ser Glu Pro Asp Gly He Ser Leu Gly Gly Ser Trp Gly Ser Trp 260 265 270 Ser Leu Pro Val Thr Val Asp Leu Pro Gly Asp Ala Val Ala Leu Gly 275 280 285

Leu Gin Cys Phe Thr Leu Asp Leu Lys Asn Val Thr Cys Gin Trp Gin 290 295 300

Gin Gin Asp His Ala Ser Ser Gin Gly Phe Phe Tyr His Ser Arg Ala 305 310 315 320

Arg Cys Cys Pro Arg Asp Arg Tyr Pro He Trp Glu Asn Cys Glu Glu 325 330 335

Glu Glu Lys Thr Asn ^" Pro Gly Leu Gin Thr Pro Gin Phe Ser Arg Cys

340 345 350 His Phe Lys Ser Arg Asn Asp Ser He He His He Leu Val Glu Val 355 360 365

Thr Thr Ala Pro Gly Thr Val His Ser Tyr Leu Gly Sei Pro Phe Trp 370 375 380

He His Gin Ala Val Arg Leu Pro Thr Pro Asn Leu His Trp Arg Glu 385 390 395 400

He Ser Ser Gl'y His Leu Glu Leu Glu Trp Gin His Pro Ser Ser Trp 405 410 415

Ala Ala Gin Glu Thr Cys Tyr Gin Leu Arg Tyr Thr Gly Glu Gly His 420 425 430 Gin Asp Trp Lys Val Leu Glu Pro Pro Leu Gly Ala Arg Gly Gly Thr 435 440 ^■ 445

Leu Glu Leu Arg Pro Arg Ser Arg Tyr Arg Leu Gin Leu Arg Ala Arg

450 455 460

Leu Asn Gly Pro Thr Tyr Gin Gly Pro Trp Ser Ser Trp Ser Asp Pro 465 470 475 480

Thr Arg Val Glu Thr Ala Thr Glu Thr Ala Trp He Ser Leu Val Thr 485 490 495

Ala Leu His Leu Val Leu Gly Leu Ser Ala Val Leu Gly Leu Leu Leu 500 505 510

Leu Arg Trp Gin Phe Pro Ala His Tyr Arg Arg Leu Arg His Ala Leu 515 520 525

Trp Pro Ser Leu Pro Asp Leu His Arg Val Leu Gly Gin Tyr Leu Arg 530 535 540

Asp Thr Ala Ala Leu Ser Pro Pro Lys Ala Thr Val Ser Asp Thr Cys 545 550 555 560

Glu Glu Val Glu Pro Ser Leu Leu Glu He Leu Pro Lys Ser Ser Glu 565 570 575

Arg Thr Pro Leu Pro Leu Cys Ser Ser Gin Ala Gin Met Asp Tyr Arg 580 585 590

Arg Leu Gin Pro Ser Cys Leu Gly Thr Met Pro Leu Ser Val Cys Pro

595 600 605 Pro Met Ala Glu Ser Gly Ser Cys Cys Thr Thr His He Ala Asn His 610 615 620

Ser Tyr Leu Pro Leu Ser Tyr Trp Gin Gin Pro 625 630 635

(2) INFORMATION FOR SEQ ID NO:5:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 36 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TACAAGCTTG CCGTCATCAT GCCCTCTTGG GCCCTC 36

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 33 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACTTCTAGAC TATCAAGCAG TCTCGGAGCT GGA 33

(2) INFORMATION FOR SEQ ID NO:7:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1523 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: unknown

(D) TOPOLOGY: unknown (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS (B) LOCATION: 69..1514

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGATCCTCTA GAGCGGCCGC TAAGGCAGGC ACACAGTGCC GGAGAAGATG CCCTCTTGGG 60

CCCTCTTC ATG GTC ACC TCC TGC CTC CTC TTG GCC CTT CCA AAC CAG GCA 110 Met Val Thr Ser Cys Leu Leu Leu Ala Leu Pro Asn Gin Ala 1 5 10

CAA GTC ACC AGC CAA GAT GTC TTC TTG CTG GCC TTG GGC ACA GAG CCC 158 Gin Val Thr Ser Gin Asp Val Phe Leu Leu Ala Leu Gly Thr Glu Pro ^' 15 20 25 30 CTG AAC TGC TTC TCC CAA ACA TTT GAG GAC CTC ACC TGC TTC TGG GAT 206 Leu Asn Cys Phe Ser Gin Thr Phe Glu Asp Leu Thr Cys Phe Trp Asp 35 40 45

GAG GAA GAG GCA GCA CCC AGT GGG ACA TAC CAG CTG CTG TAT GCC TAC 254 Glu Glu Glu Ala Ala Pro Ser Gly Thr Tyr Gin Leu Leu Tyr Ala Tyr

50 55 60

CGA GGA GAG AAG CCC CGT GCA TGC CCC CTG TAT TCC CAG AGT GTG CCC 302 Arg Gly Glu Lys Pro Arg Ala Cys Pro Leu Tyr Ser Gin Ser Val Pro 65 70 75

ACC TTT GGA ACC CGG TAT GTG TGC CAG TTT CCA GCC CAG GAT GAA GTG 350 Thr Phe Gly Thr Arg Tyr Val Cys Gin Phe Pro Ala Gin Asp Glu Val 80 85 90 CGC CTC TTC TTT CCG CTG CAC CTC TGG GTG AAG AAT GTG TCC CTC AAC 398 Arg Leu Phe Phe Pro Leu His Leu Trp Val Lys Asn Val Ser Leu Asn 95 100 105 110

CAG ACT TTG ATC CAG CGG GTG CTG TTT GTG GAT AGT GTG GGC CTG CCA 446 Gin Thr Leu He Gin Arg Val Leu Phe Val Asp Ser Val Gly Leu Pro

115 120 125

GCT CCC CCC AGG GTC ATC AAG GCC AGG GGT GGG AGC CAA CCA GGG GAA 494 Ala Pro Pro Arg Val He Lys Ala Arg Gly Gly Ser Gin Pro Gly Glu 130 135 140

CTT CAG ATC CAC TGG GAG GCC CCT GCT CCT GAA ATC AGT GAC TTC CTG 542 Leu Gin He His Trp Glu Ala Pro Ala Pro Glu He Ser Asp Phe Leu 145 150 155

AGG CAT GAA CTC CGC TAT GGC CCC ACG GAT TCC AGC AAC GCC ACT GCC 590 Arg His Glu Leu Arg Tyr Gly Pro Thr Asp Ser Ser Asn Ala Thr Ala 160 165 170 CCC TCC GTC ATT CAG CTG CTC TCC ACA GAA ACC TGC TGC CCC ACT TTG 638 Pro Ser Val He Gin Leu Leu Ser Thr Glu Thr Cys Cys Pro Thr Leu 175 180 185 190

TGG ATG CCG AAC CCA GTC CCT GTT CTT GAC CAG CCT CCG TGT GTT CAT 686 Trp Met Pro Asn Pro Val Pro Val Leu Asp Gin Pro Pro Cys Val His

195 200 205

CCG ACA GCA TCC CAA CCG CAT GGA CCA GTG AGG ACC TCC CCA GCT GGA 734 Pro Thr Ala Ser Gin Pro His Gly Pro Val Arg Thr Ser Pro Ala Gly 210 215 220

GAA GCT CCA TTT CTG ACA GTG AAG GGT GGA AGC TGT CTC GTC TCA GGC 782

Glu Ala Pro Phe Leu Thr Val Lys Gly Gly Ser Cys Leu Val Ser Gly

225 230 235

CTC CAG GCT AGC AAA TCC TAC TGG CTC CAG CTA CGC AGC CAA CCC GAC 830

Leu Gin Ala Ser Lys Ser Tyr Trp Leu Gin Leu Arg Ser Gin Pro Asp

240 245 250 GGG GTC TCT CTT CGT GGC TCC TGG GGA CCC TGG TCC TTC CCT GTG ACT 878 Gly Val Ser Leu Arg Gly Ser Trp Gly Pro Trp Ser Phe Pro Val Thr 255 260 265 270

GTG GAT CTT CCA GGA GAT GCA GTG ACA ATT GGA CTT CAG TGC TTT ACC 926 Val Asp Leu Pro Gly Asp Ala Val Thr He Gly Leu Gin Cys Phe Thr

275 280 285

TTG GAT CTG AAG ATG GTC ACC TGC CAG TGG CAG CAA CAA GAC CGC ACT 974 Leu Asp Leu Lys Met Val Thr Cys Gin Trp Gin Gin Gin Asp Arg Thr 290 295 300

AGC TCC CAA GGC TTC TTC CGT CAC AGC AGG ACG AGG TGC TGC CCC ACA 1022 Ser Ser Gin Gly Phe Phe Arg His Ser Arg Thr Arg Cys Cys Pro Thr 305 310 315

GAC AGG GAC CCC ACC TGG GAG AAA TGT GAA GAG GAG GAA CCG CGT CCA 1070 Asp Arg Asp Pro Thr Trp Glu Lys Cys Glu Glu Glu Glu Pro Arg Pro 320 325 330

GGA TCA CAG CCC GCT CTC GTC TCC CGC TGC CAC TTC AAG TCA CGA AAT 1118 Gly Ser Gin Pro Ala Leu Val Ser Arg Cys His Phe Lys Ser Arg Asn

335 340 345 350

GAC AGT GTT ATT CAC ATC CTT GTA GAG GTG ACC ACA GCG CAA GGT GCC 1166 Asp Ser Val He His He Leu Val Glu Val Thr Thr Ala Gin Gly Ala 355 360 365

GTT CAC AGC TAC CTG GGC TCC CCT TTT TGG ATC CAC CAG GCT GTG CTC 1214 Val His Ser Tyr Leu Gly Ser Pro Phe Trp He His Gin Ala Val Leu 370 375 380

CTT CCC ACC CCG AGC CTG CAC TGG AGG GAG GTC TCA AGT GGA AGG CTG 1262

Leu Pro Thr Pro Ser Leu His Trp Arg Glu Val Ser Ser Gly Arg Leu

385 390 395 GAG TTG GAG TGG CAG CAC CAG TCA TCT TGG GCA GCT CAA GAG ACC TGC 1310 Glu Leu Glu Trp Gin His Gin Ser Ser Trp Ala Ala Gin Glu Thr Cys 400 405 410

TAC CAG CTC CGG TAC ACG GGA GAA GGC CGT GAG GAC TGG AAG GTG CTG 1358 Tyr Gin Leu Arg Tyr Thr Gly Glu Gly Arg Glu Asp Trp Lys Val Leu 415 420 425 430

GAG CCA TCT CTC GGT GCC CGG GGA GGG ACC CTA GAG CTG CGC CCC CGA 1406 Glu Pro Ser Leu Gly Ala Arg Gly Gly Thr Leu Glu Leu Arg Pro Arg 435 440 445

GCT CGC TAC AGC TTG CAG CTG CGT GCC AGG CTC AAC GGC CCC ACC TAC 1454

Ala Arg Tyr Ser Leu Gin Leu Arg Ala Arg Leu Asn Gly Pro Thr Tyr 450 455 460

CAA GGT CCC TGG AGC GCC TGG TCT CCC CCA GCT AGG GTG TCC ACG GGC 1502

Gin Gly Pro Trp Ser Ala Trp Ser Pro Pro Ala Arg Val Ser Thr Gly 465 470 475 TCC GAG ACT GCT TGAGTCGAC 1523

Ser Glu Thr Ala 480

(2) INFORMATION FOR SEQ ID NO:8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 482 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 :

Met Val Thr Ser Cys Leu Leu Leu Ala Leu Pro Asn Gin Ala Gin Val 1 5 10 15

Thr Ser Gin Asp Val Phe Leu Leu Ala Leu Gly Thr Glu Pro Leu Asn 20 25 30 Cys Phe Ser Gin Thr Phe Glu Asp Leu Thr Cys Phe Trp Asp Glu Glu 35 40 45

Glu Ala Ala Pro Ser Gly Thr Tyr Gin Leu Leu Tyr Ala Tyr Arg Gly 50 55 60

Glu Lys Pro Arg Ala Cys Pro Leu Tyr Ser Gin Ser Val Pro Thr Phe 65 70 75 80

Gly Thr Arg Tyr Val Cys Gin Phe Pro Ala Gin Asp Glu Val Arg Leu 85 90 95

Phe Phe Pro Leu His Leu Trp Val Lys Asn Val Ser Leu Asn Gin Thr

100 105 110 Leu He Gin Arg Val Leu Phe Val Asp Ser Val Gly Leu Pro Ala Pro 115 120 125

Pro Arg Val He Lys Ala Arg Gly Gly Ser Gin Pro Gly Glu Leu Gin 130 135 140

He His Trp Glu Ala Pro Ala Pro Glu He Ser Asp Phe Leu Arg His 145 150 155 160

Glu Leu Arg Tyr Gly Pro Thr Asp Ser Ser Asn Ala Thr Ala Pro Ser 165 170 175

Val He Gin Leu Leu Ser Thr Glu Thr Cys Cys Pro Thr Leu Trp Met 180 185 190 Pro Asn Pro Val Pro Val Leu Asp Gin Pro Pro Cys Val His Pro Thr 195 200 205

Ala Ser Gin Pro His Gly Pro Val Arg Thr Ser Pro Ala Gly Glu Ala 210 215 220

Pro Phe Leu Thr Val Lys Gly Gly Ser Cys Leu Val Ser Gly Leu Gin

225 230 235 240

Ala Ser Lys Ser Tyr Trp Leu Gin Leu Arg Ser Gin Pro Asp Gly Val 245 250 255

Ser Leu Arg Gly Ser Trp Gly Pro Trp Ser Phe Pro Val Thr Val Asp

260 265 270 Leu Pro Gly Asp Ala Val Thr He Gly Leu Gin Cys Phe Thr Leu Asp

275 280 285

Leu Lys Met Val Thr Cys Gin Trp Gin Gin Gin Asp Arg Thr Ser Ser

290 295 300

Gin Gly Phe Phe Arg His Ser Arg Thr Arg Cys Cys Pro Thr Asp Arg 305 310 315 320

Asp Pro Thr Trp Glu Lys Cys Glu Glu Glu Glu Pro Arg Pro Gly Ser 325 330 335 Gin Pro Ala Leu Val Ser Arg Cys His Phe Lys Ser Arg Asn Asp Ser

340 345 350

Val He His He Leu Val Glu Val Thr Thr Ala Gin Gly Ala Val His 355 360 365

Ser Tyr Leu Gly Ser Pro Phe Trp He His Gin Ala Val Leu Leu Pro 370 375 380

Thr Pro Ser Leu His Trp Arg Glu Val Ser Ser Gly Arg Leu Glu Leu 385 390 395 400

Glu Trp Gin His Gin Ser Ser Trp Ala Ala Gin Glu Thr Cys Tyr Gin

405 410 415 Leu Arg Tyr Thr Gly Glu Gly Arg Glu Asp Trp Lys Val Leu Glu Pro

420 425 430

Ser Leu Gly Ala Arg Gly Gly Thr Leu Glu Leu Arg Pro Arg Ala Arg 435 440 445

Tyr Ser Leu Gin Leu Arg Ala Arg Leu Asn Gly Pro Thr Tyr Gin Gly 450 455 460

Pro Trp Ser Ala Trp Ser Pro Pro Ala Arg Val Ser Thr Gly Ser Glu 465 470 475 480

Thr Ala