AU770157B2 - Use of semaphorin for modulation of cellular efflux - Google Patents

Description

WO 01/18044 PCT/US00/24560 SEMAPHORIN MODULATION OF CELLULAR EFFLUX This application claims priority to United States provisional patent application serial number 60/152,914 filed September 8, 1999, now abandoned; United States provisional patent application serial number 60/156,257, filed September 27, 1999, now abandoned; and United States provisional patent application serial number 60/173,906 filed December 29, 1999, now abandoned.

I. FIELD OF THE INVENTION The present invention relates to compositions and methods useful in manipulating cellular efflux mechanisms resulting in multiple drug resistance (MDR). More specifically, the present invention relates to the use of semaphorin or semaphorin receptor polypeptides, as well as polynucleotides encoding these polypeptides, to modulate cellular efflux or the MDR phenotype of cells.

II. BACKGROUND OF THE INVENTION In response to unavoidable continuous exposure to a frequently hostile environment, cells have developed a multitude of mechanisms to prevent entry or to accelerate exit of noxious substances from the intra-cellular space. This "cellular Darwinism" is accepted as a basic tool of survival, but, once applied by targeted cells to cytotoxic drugs, the phenomenon interferes with the effectiveness of chemotherapies for an array of diseases such as cancer and HIV. As the result of a wide spectrum of highly effective systems, drug resistance, whatever its source, is a prevalent cause for chemotherapeutic failure.

When cellular resistance to one drug results in resistance to a wide array of chemical agents, including those that are not related to the substance originally inducing the resistance, the cell is regarded as having developed multidrug resistance, or MDR. Thus, MDR is a cellular phenomenon characterized by resistance of the cell to cytotoxic substances. Generally, MDR develops in response to a specific cytotoxic substance, but then confers resistance to an array of cytotoxic substances or conditions. Cells that have developed MDR are considered MDR phenotypic cells, and are further described as those cells that have an increased ability, relative to non-MDR cells, to survive in the presence of cytotoxic substances or cytotoxic conditions. The increased survival rates of MDR phenotypic cells is characteristically due to an increased cellular capacity to efflux or expel from the cell substances that are either cytotoxic in themselves, or are present in the cell in cytotoxic amounts, thereby creating a cytotoxic condition for the cell. In an attempt to understand and control MDR, many investigators have studied the various mechanisms thought to drive it. See Kellen, Alternative Mechanisms of Multidrue Resistance in Cancer, (1995). MDR phenotypes of cancer or other cells may arise as a result of MDR proteins, or MDR-like proteins, or various other mechanisms involving efflux pumps. Cellular efflux pumps involved in the development of MDR phenotypic cells include those that are able to efflux molecules of many different sizes and compositions, as well as protons or 'i WO 01/18044 PCT/US00/24560 chloride ions. For example, MDR protein pumps include the proteins MDR-1 and MDR-2, which are each considered to be a P-glycoprotein and the human multiple drug resistance associated protein designated "MRP" (see, Zaman, et al., 1994). These and other MDR proteins are transmembrane efflux pumps that, based on studies in the mouse, are believed to be important in removing toxins from the cell.

Various assays that have been developed to allow the study of exchange of molecules across membranes are employed in the study of MDR proteins. For example, many lipophilic, cationic dyes have been described that allow one to follow changes in membrane potential, or changes in intracellular pH. One such dye, Rhodamine 123 (Rhl23) was frequently used by hematologists to measure mitochondrial membrane potential, and has been described as a substrate for MDR proteins.

Kim et.al., (1998). Consistent with the reported transport of protons, expression of the MDR protein Pgp has been associated with a significant elevation of intracellular pH (Weisburg et al, 1999).

Further, MDR phenotypes are reported to arise in some cell types as a result of alterations in the acidification (pH) of intracellular organelles and compartments, such as the trans-golgi network and the endocytic pathway (see, Altan, et al., Altan, N et al., Chen, Y et al., Schindler, et al.).

One mechanism for controlling the pH of intracellular compartments is by cellular pumps that operate to move protons, or negatively charged ions like chloride ions, across membranes. Such cellular pumps are implicated in certain diseases. For example, unregulated activity of a chloride pump is known to be at least partially responsible for the development of cystic fibrosis resulting from a genetic defect. Alternatively, growth factors are theorized to play a non-efflux-related role in MDR.

For example, semaphorins have been postulated to function as growth factors, and thereby exert an effect on cells that may contribute to the development of drug resistance (Yamada, et al.).

In light of the various relationships between cellular efflux pumps and MDR, the ability to control such efflux pumps would provide the ability to promote or suppress the development of MDR in cells. Accordingly, investigation into MDR mechanisms, and various methods for controlling MDR via control of cellular efflux mechanisms is ongoing.

m. SUMMARY OF THE INVENTION- The present invention teaches the use of semaphorin or semaphorin receptor polypeptides to modulate the activity of cellular efflux pumps. The present invention further teaches that semaphorin or semaphorin receptor polypeptides can be used to specifically activate or inhibit cellular efflux pumps and therefore may induce or inhibit the development of multiple drug resistant cells. The present invention further provides compositions and methods for the treatment of neoplasms, autoimmune or immune-deficiency disorders such as HIV, and other cellular-efflux-related disease states.

WO 01/18044 PCTIUS00/24560 The present invention specifically contemplates that any semaphorin polypeptide, or active fragment of a semaphorin polypeptide, may be used in the disclosed compositions and methods.

Exemplary semaphorins include, for example and without limitation: AHV Sema; A39R; Sema I, including G-sema I and D-sema-I; Sema II; Sema III; Sema IV; DC Sema; CD100; Z SMF-7; Sema A; Sema B; Sema C; Sema D; Sema E; Sema H; Sema L; Sema W and Sema Y. Additionally, useful fragments of any semaphorin, such as the sema domain or the active domain may also be used according to the present invention. For additional semaphorins that can be used in the presently disclosed compositions and methods, see Bamberg, et.al. Cell, 97:551 and United States Patent No.

5,935,865 to Goodman et al. In alternative embodiments of the present invention, such as in "gene therapeutics," nucleic acid sequences encoding any of these semaphorins or their fragments can be used.

Similarly, preferred semaphorin receptor polypeptides for use in the presently disclosed compositions and methods include those semaphorin receptors known as plexins, as well as their complements, variants and useful fragments such as soluble portions of the receptors, fragments including the sema domain of the plexins, and fragments including the active sites of the plexins. A particularly preferred plexin for use according to the present invention is the Viral-Encoded Semaphorin Receptor ("VESPR"), as well as complements, variants, and soluble fragments thereof.

Particularly preferred polypeptide sequences include the polypeptide sequence of SEQ ID NO:2.

Additionally, useful soluble forms of the VESPR polypeptide include those segments of the polypeptide comprising a portion of the extracellular domain of the receptor. An example of a soluble VESPR polypeptide includes amino acids 1-944 of SEQ ID NO:2. In addition, truncated soluble VESPR proteins comprising less that the entire extracellular domain are included in the invention, e.g., amino acids 35-944. Also encompassed within the present invention are the nucleic acid sequences encoding such useful VESPR polypeptides and polypeptide fragments. Particularly preferred nucleic acid sequences include the polynucleotide sequence of SEQ ID NO: 1; and those segments of SEQ ID NO:1 that encode the soluble fragments of VESPR outlined above. The VESPR, its useful fragments, complements, variants, and combinations, such as fusion proteins as well as the nucleic acid sequences encoding these polypeptides are described in co-pending application SN 08/958,598 (specifically incorporated herein by reference, in its entirety). In embodiments of the present invention employing nucleic acid sequences, such as in "gene therapeutics," nucleic acid sequences encoding any of these semaphorin receptor polypeptides or their fragments can be used.

In a preferred embodiment, the present invention provides a pharmaceutical composition for the treatment of MDR phenotypic cells. This composition comprises an amount of a semaphorin or a semaphorin receptor polypeptide such that administration of the composition is effective to modulate the MDR phenotype of the target cells. Alternatively, in another aspect of the invention, the WO 01/18044 PCT/US00/24560 composition further includes an amount of an expression vector including a nucleic acid sequence encoding a semaphorin, a semaphorin receptor, or a useful fragment of a semaphorin or semaphorin receptor, such that administration of the composition is effective to modulate the MDR phenotype of the target cell. This modulation may be to either promote or inhibit the development of multiple drug resistant cells.

An alternative embodiment of the present invention provides another pharmaceutical composition for the treatment of MDR phenotypic cells. In this aspect, the presently disclosed composition includes an amount of an agonist or antagonist for a semaphorin or a semaphorin receptor, such that administration of the composition is effective to promote or inhibit the development of MDR phenotype. Exemplary agonists or antagonists for semaphorins or semaphorin receptors include antibodies, such as, for example, either polyclonal or monoclonal antibodies, antigens and small molecules.

For example, a composition of the present invention can use a semaphorin antagonist, in the form of a soluble semaphorin receptor for example, to inhibit induction or activation of cellular efflux pumps. Use of such a composition allows one to decrease the ability of a cell to expel agents crossing the cell membrane, such as cytotoxic therapeutic agents. Alternatively or additionally, a composition of the present invention can include an antibody to a semaphorin receptor such as VESPR, which can function as either an antagonist or an agonist, or a small molecule agonist of a semaphorin receptor such as VESPR can be used.

In another embodiment, the present invention provides a pharmaceutical composition, for the treatment of cellular efflux-related disease states. In this aspect, the composition includes an amount of a semaphorin or semaphorin receptor such that administration of the composition is effective to modulate cellular efflux. Alternatively, in this aspect of the invention, the composition includes an amount of an expression vector including a nucleic acid sequence encoding a semaphorin, a semaphorin receptor, or encoding a useful fragment of a semaphorin or semaphorin receptor, such that administration of the composition is effective to modulate cellular efflux of the target cells. The active polypeptide or nucleic acid sequences of the composition used in this aspect of the invention may function to activate or up-regulate, or to inhibit or down-regulate, cellular efflux.

In an alternative embodiment, the present invention provides another composition for the treatment of cellular efflux-related disease states. In this embodiment, the disclosed composition includes an amount of an agonist or antagonist of a semaphorin or semaphorin receptor, such that administration of the composition is effective in activating or inhibiting cellular efflux in the target cell. Exemplary agonists or antagonists for semaphorins or semaphorin receptors include antibodies, such as, for example, either polyclonal or monoclonal antibodies; antigens and small molecules.

i WO 01118044 PCTIUS00/24560 In another aspect, the present invention provides a method of modulating cellular efflux by administering to a cell an effective amount of a composition including a semaphorin or semaphorin receptor polypeptide such that cellular efflux is activated or inhibited. Alternatively, the present invention provides a method of modulating cellular efflux comprising administering to a cell, via an appropriate vector, an effective amount of a polynucleotide encoding a semaphorin, a semaphorin receptor, or a useful fragment of a semaphorin or semaphorin receptor, such that cellular efflux is activated or inhibited. Additionally, the presently disclosed methods of modulating cellular efflux, may comprise administering to a cell an effective amount of an agonist or antagonist of a semaphorin or semaphorin receptor such that cellular efflux is activated or inhibited. Exemplary useful agonists or antagonists include antibodies such as, for example, monoclonal or polyclonal antibodies, an antigen, or a small molecule. In a particularly preferred embodiment, the antibody used is an antibody to

VESPR.

Pharmaceutical compositions and methods of the presently disclosed invention may be useful in the treatment of cellular efflux-related disease states such as multiple drug resistance; cancers, or other neoplastic diseases such as tumors, leukemia, lymphoma or other localized or metastatic conditions characterized by an abnormal proliferation of cells, generally due to cells continuing to replicate after the stimuli that initiated growth has ceased; cystic fibrosis arising from the treatment of a cell or group of cells with cytotoxic agents; auto-immune disorders; or acquired or genetically-based immunodeficiency disorders such as that resulting from the human immunodeficiency virus (HIV).

Formulation of any of the presently disclosed compositions for administration according to the disclosed methods can be done in any manner known to those of skill in the art. Such formulations will vary according to variables such as, for example, the needs of the formulator, the intended route of administration, the targeted disease or tissue, and the subject being treated. Specifically, unit doses may be formulated in multi-dose containers including additives such as a carrier, other excipients, and a preservative component.

The disclosed compositions may be formulated in a variety of concentrations in various vial sizes for various administration dosages. The presently disclosed compositions may also be in virtually any form including an aqueous solution, a suspension, a lyophilized form that may be reconstituted when appropriate, a gel, an aerosol, or any other form or state convenient for administration to treat the described disorders. The compositions as described herein may be formulated so that they are contained in a vial, bottle, tube, syringe, inhaler, transdermal patch, capsule or other container for single or multiple administrations.

In alternative embodiments, the presently disclosed compositions are formulated with or administered in conjunction with additional active agents such as chemotherapeutic agents, immune suppressants or radiation therapy. For example, agents that may be useful to co-formulate or WO 01/18044 PCT/US00/24560 administer in conjunction with the disclosed compositions include virtually any chemotherapeutic or sensitizing agent such as cyclosporin, FK506, taxotere, doxorubicin, cis-platin, tamoxifen, iphosphamide, or methotrexate, or variants of any of these compounds. Alternatively or additionally, the presently disclosed compositions may be further co-administered with an immune suppressant, such as a cytokine, IL-4, IL-12,, GM-CSF, G-CSF, M-CSF, a-interferon, P-interferon, or yinterferon. The additional agents may be co-administered simultaneously or sequentially relative to the disclosed compositions and methods.

In another aspect, the present invention provides various assays and screening methods to identify substances that may be used to influence the MDR phenotype of a cell. For example, the present invention provides a method of detecting the ability of a test compound to affect the MDR phenotype of a cell, in which the following steps are used: contacting a first cell with a test compound and a semaphorin or a semaphorin receptor, in the presence of a cytotoxic agent; (2) measuring the rate of death of the first cell; observing the rate of death of a control cell in the absence of the test compound; and comparing the rate of death of the first cell to the rate of death of the control cell. Upon comparison, a difference in the rate of cell death of the first cell relative to the control cell indicates that the test compound is an effector of MDR phenotype. In this manner, the effector can be identified as a substance that either promotes development of MDR phenotype or inhibits development of MDR phenotype. The affector can then be used therapeutically.

Alternatively, the test compound may itself be a semaphorin or semaphorin receptor or fragment or antagonist or agonist thereof.

This method can be performed with a cytotoxic or sensitizing agent such as, for example, tamoxifen, cisplatin, doxorubicin, radiation, methotrexate, cyclosporin, taxotere, FK506, or iphosphamide. Further, as with all compositions and methods of the present invention, the semaphorin or semaphorin receptor used in this method can be any known semaphorin or receptor polypeptide or useful fragment thereof, such as a fragment comprising the sema domain or the active domain of a semaphorin or semaphorin receptor. Additionally or alternatively, the presently disclosed method can be performed with any known semaphorin or semaphorin receptor, or fragment thereof being the test compound, or with an antibody to VESPR as the test compound.

In another aspect, the present invention provides a method of detecting the ability of a test compound to effect the MDR phenotype of a cell by modulating cellular efflux in the cell. Such a method would involve, for example, the following steps: contacting a first cell with a test compound and a semaphorin or semaphorin receptor, in the presence of a dye; measuring the net rate of influx of dye into the first cell; observing the net rate of influx of dye into a control cell, in the absence of test compound comprising a semaphorin or semaphorin receptor, under otherwise identical conditions; and comparing the net rate influx of dye into the first cell to the net rate of WO 01/18044 PCT/US00/24560 influx of dye into the control cell. Upon comparison, a difference in the net rate of influx of dye into the first cell relative to the control cell indicates that the test compound is an effector of cellular efflux.

In this manner, the effector can be identified as a substance that either promotes cellular efflux or inhibits cellular efflux and then can be used therapeutically. Alternatively, the test compound may itself be a semaphorin or semaphorin receptor or fragment or antagonist or agonist thereof.

Any dye may be used in the assays of the present invention. The dyes useful in such methods may be characterized by, for example, one or more of the following properties: lipophilic, cationic, fluorescent, and radioactive. Alternatively or additionally, the dye used in such methods can be a slow dye, a fast dye, acridine orange, BODIPY ceramide, SNARF-dextran, FITC-transferrin or BODIPYtransferrin.

As with all compositions and methods of the present invention, the semaphorin or semaphorin receptor used in this method can be any known semaphorin or semaphorin receptor polypeptide or useful fragment thereof, such as a fragment comprising the sema domain or the active domain of a semaphorin or semaphorin receptor. Additionally or alternatively, the presently disclosed method can be performed with any known semaphorin or semaphorin receptor, or fragment thereof being the test compound, or with an antibody to VESPR as the test compound.

In yet another aspect, the present invention provides pharmaceutical compositions and methods for the regulation of cellular-efflux, or MDR phenotype, by using the agent identified by the assays described herein. In this aspect of the invention, the modulating agent is effective to either inhibit or activate cellular efflux or development of drug resistance in a target cell.

IV. DETAILED DESCRIPTION OF THE INVENTION Contrary to the results of Yamada et al., who postulate that semaphorins function analogously to growth factors and may be involved in non-MDR drug resistance, the present invention teaches that semaphorins and semaphorin receptors can be used to influence the function of cellular efflux pumps in a variety of ways, including activation, inhibition, and promotion of stasis of the pumps and can be used to regulate MDR. The invention also teaches that, depending upon the specific semaphorin/receptor interaction, this influence can be inhibitory, and the capacity of a cell to eliminate cellular contents can be reduced, or the influence can be to promote cellular efflux and thereby facilitate expulsion of cellular contents. Accordingly, depending upon the effect, the disclosed semaphorin and semaphorin receptor compositions and methods are also useful: to increase vulnerability or sensitivity of a cell to cytotoxic agents and thereby promote drug-induced cell death; in identification or design ofsemaphorin or semaphorin receptor antagonists or agonists that might increase the sensitivity of a cell to a cytotoxic agent; to promote cellular resistance to cytotoxic agents; or in identification of semaphorin or semaphorin receptor agonists or antagonists that can be administered to cells to promote their resistance to various cytotoxic substances.

WO 01/18044 PCT/US00/24560 A. SEMAPHORIN AND SEMAPHORIN RECEPTOR POLYPEPTIDES The terms "semaphorin" and "semaphorin polypeptide" are used interchangeably in the present invention. Semaphorins include proteins of the Semaphorin family and are either secreted or membrane-bound. Semaphorins have a well-conserved extracellular semaphorin (sema) domain.

Generally, the sema domain is approximately 500 residues, but viral semaphorins themselves are only approximately 440 to 441 amino acids in length. It has been hypothesized that a 70 amino acid region with the sema domain is the active domain for semaphorin influence on certain cellular activities. See Koppel, et al. (1997). However it is not clear that this same region is the active site for all semaphorin activity. Accordingly, the present invention specifically contemplates the use of full-length semaphorin polypeptides, variants of these, and useful fragments of semaphorin polypeptides.

Specific semaphorins and semaphorin fragments that are useful according to the present invention include, for example, the following semaphorins: AHV Sema; A39R; Sema I, including G-sema I and D-sema-I; Sema II; Sema III; Sema IV; DC Sema; CD100; Z SMF-7; Sema A; Sema B; Sema C; Sema D; Sema E; Sema H; Sema L; Sema W and Sema Y. Additionally, useful fragments of any semaphorin, such as the sema domain or the active domain may also be used according to the present invention. For additional semaphorins that can be used in the presently disclosed compositions and methods, see Bamberg, et.al. Cell, 97:551 and United States Patent No. 5,935,865 to Goodman et al.

Nucleic acid sequences encoding the semaphorins or semaphorin fragments of the present invention, are also specifically contemplated to be useful in the disclosed compositions and methods.

"Semaphorin receptors" or "semaphorin receptor polypeptides" of the present invention are members of the Plexin family of semaphorin receptors. Plexins are membrane-bound polypeptides.

Plexins contain a "sema" domain that is related to the sema domain of semaphorins themselves, part of which constitutes a series of two or three cystein repeat sequences in the extracellular domain of plexins. Plexins are distinct from semaphorins, however, in a variety of respects. For example, in their intracellular domain, plexins are strongly homologous throughout the family of plexins, and contain well-conserved amino acid motifs that are not found in semaphorins.

Semaphorin receptors of the present invention are those plexin polypeptide sequences that can interact with a semaphorin or a semaphorin fragment, to influence cellular efflux or development of MDR phenotype in a cell Exemplary semaphorin receptor polypeptides include full-length plexin receptor polypeptides as well as homologues or fragments, such as the soluble extra cellular domain or the sema domain of such plexin receptor polypeptides. Preferred semaphorin receptor polypeptides include the Viral Encoded Semaphorin Receptor (VESPR) or fragments thereof. As used herein, the term VESPR or VESPR polypeptide refers to any polypeptide functioning as a receptor for viral semaphorins, for human homologues to viral semaphorins, or for human semaphorins.

I WO 01/18044 PCT/US00/24560 Additionally, useful soluble forms of the VESPR polypeptide include those segments of the polypeptide comprising a portion of the extracellular domain of the receptor. An example of a soluble VESPR polypeptide includes amino acids 1-944 of SEQ ID NO:2. In addition, truncated soluble VESPR proteins comprising less that the entire extracellular domain are included in the invention, e.g., amino acids 35-944. Also encompassed within the present invention are the nucleic acid sequences encoding such useful VESPR polypeptides and polypeptide fragments. An exemplary Plexin receptor is the Viral Encoded Semaphorin Protein Receptor "VESPR," (described in copending patent application serial number 08/958,598). Specifically the amino acid sequence of SEQ ID NO:2 is useful as a semaphorin receptor polypeptide in the presently disclosed compositions and methods, as are the homologues and variants of polypeptides of SEQ ID NO:2. Nucleic acid sequences encoding the semaphorin receptors or receptor fragments are also within the scope of the presently disclosed compositions and methods. Particularly preferred nucleic acid sequences include the polynucleotide sequence of SEQ ID NO:1; and those segments of SEQ ID NO: I that encode the soluble fragments of VESPR outlined above.

The semaphorin or semaphorin receptor polypeptides of the invention may be membrane bound or they may be secreted and thus soluble. Soluble polypeptides are capable of being secreted from the cells in which they are expressed. In general, soluble polypeptides may be identified (and distinguished from non-soluble membrane-bound counterparts) by separating intact cells which express the desired polypeptide from the culture medium, by centrifugation, and assaying the medium (supematant) for the presence of the desired polypeptide. The presence of polypeptide in the medium indicates that the polypeptide was secreted from the cells and thus is a soluble form of the protein.

In one embodiment, the soluble polypeptides and fragments thereof comprise all or part of the extracellular domain, but lack the transmembrane region that would cause retention of the polypeptide on a cell membrane. A soluble polypeptide may include the cytoplasmic domain, or a portion thereof, as long as the polypeptide is secreted from the cell in which it is produced.

In general, the use of soluble forms is advantageous for certain applications. Purification of the polypeptides from recombinant host cells is facilitated, since the soluble polypeptides are secreted from the cells. Further, soluble polypeptides are generally more suitable for intravenous administration.

The invention also provides polypeptides and fragments of the extracellular domain that retain a desired biological activity. Particular embodiments are directed to polypeptide fragments that retain the ability to interact with the semaphorin receptor or ligand to influence cellular efflux or the MDR phenotype of a cell. Such a fragment may be a soluble polypeptide, as described above. In another embodiment, the polypeptides and ragments advantageously include regions that are conserved in the iI"^ WO 01/18044 PCTIUS00/24560 semaphorin family, in the case of semaphorins; or regions that are conserved in the plexin family in the case of the semaphorin receptors; or include the sema domain of either.

Also provided herein are polypeptide fragments comprising at least 20, or at least contiguous amino acids of the sequence of SEQ ID NO:2. Fragments derived from the cytoplasmic domain find use in studies of signal transduction, and in regulating cellular processes associated with transduction of biological signals. Polypeptide fragments also may be employed as immunogens, in generating antibodies.

Naturally occurring variants as well as derived variants of the polypeptides and fragments are provided herein. Variants may exhibit amino acid sequences that are at least 80% identical. Also contemplated are embodiments in which a polypeptide or fragment comprises an amino acid sequence that is at least 90% identical, at least 95% identical, at least 98% identical, at least 99% identical, or at least 99.9% identical to the preferred polypeptide or fragment thereof. Percent identity can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two protein sequences can be determined by comparing sequence information using the GAP computer program, based on the algorithm of Needleman and Wunsch Mol. Bio. 48:443, 1970) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters for the GAP program include: a scoring matrix, blosum62, as described by Henikoff and Henikoff (Proc. Natl. Acad. Sci. USA 89:10915, 1992); a gap weight of 12; a gap length weight of 4; and no penalty for end gaps. Other programs used by one skilled in the art of sequence comparison may also be used.

The variants of the invention include, for example, those that result from alternate mRNA splicing events or from proteolytic cleavage. Alternate splicing ofmRNA may, for example, yield a truncated but biologically active protein, such as a naturally occurring soluble form of the protein.

Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the protein (generally from 1-5 terminal amino acids). Proteins in which differences in amino acid sequence are attributable to genetic polymorphism (allelic variation among individuals producing the protein) are also contemplated herein.

Additional variants within the scope of the invention include polypeptides that may be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. Covalent derivatives may be prepared by linking the chemical moieties to functional groups on amino acid side chains or at the N-terminus or C-terminus of a polypeptide. Conjugates comprising diagnostic (detectable) or therapeutic agents attached thereto are contemplated herein, as discussed in more detail below.

i~ ~I WO 01/18044 PCT/US00/24560 Other derivatives include covalent or aggregative conjugates of the polypeptides with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. Examples of fusion proteins are discussed below in connection with oligomers. Further, fusion proteins can comprise peptides added to facilitate purification and identification. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Patent No.

5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988. One such peptide is the FLAG peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein. A murine hybridoma designated 4E11 produces a monoclonal antibody that binds the FLAG* peptide in the presence of certain divalent metal cations, as described in U.S. Patent 5,011,912, hereby incorporated by reference. The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259. Monoclonal antibodies that bind the FLAG* peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Connecticut.

Among the variant polypeptides provided herein are variants of native polypeptides that retain the native biological activity or the substantial equivalent thereof. One example is a variant that binds with essentially the same binding affinity as does the native form. Binding affinity can be measured by conventional procedures, as described in U.S. Patent No. 5,512,457 and as set forth below.

Variants include polypeptides that are substantially homologous to the native form, but which have an amino acid sequence different from that of the native form because of one or more deletions, insertions or substitutions. Particular embodiments include, but are not limited to, polypeptides that comprise from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a native sequence.

A given amino acid may be replaced, for example, by a residue having similar physiochemical characteristics. Examples of such conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one.another; substitutions of one polar residue for another, such as between Lys and Arg, Glu and Asp, or Gin and Asn; or substitutions of one aromatic residue for another, such as Phe, Trp, or Tyr for one another. Other conservative substitutions, e.g., involving substitutions of entire regions having similar hydrophobicity characteristics, are well known.

Similarly, the DNAs of the invention include variants that differ from a native DNA sequence because of one or more deletions, insertions or substitutions, but that encode a biologically active polypeptide.

The invention further includes polypeptides of the invention with or without associated nativepattern glycosylation. Polypeptides expressed in yeast or mammalian expression systems COS-1 or COS-7 cells) can be similar to or significantly different from a native polypeptide in molecular WO 01/18044 PCT/US00/24560 weight and glycosylation pattern, depending upon the choice of expression system. Expression of polypeptides of the invention in bacterial expression systems, such as E. coli, provides nonglycosylated molecules. Further, a given preparation may include multiple differentially glycosylated species of the protein. Glycosyl groups can be removed through conventional methods, in particular those utilizing glycopeptidase. In general, glycosylated polypeptides of the invention can be incubated with a molar excess of glycopeptidase (Boehringer Mannheim).

Correspondingly, similar DNA constructs that encode various additions or substitutions of amino acid residues or sequences, or deletions of terminal or internal residues or sequences are encompassed by the invention. For example, N-glycosylation sites in the polypeptide extracellular domain can be modified to preclude glycosylation, allowing expression of a reduced carbohydrate analog in mammalian and yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Appropriate substitutions, additions, or deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues at the Asn side chain.

Alteration of a single nucleotide, chosen so that Asn is replaced by a different amino acid, for example, is sufficient to inactivate an N-glycosylation site. Alternatively, the Ser or Thr can by replaced with another amino acid, such as Ala. Known procedures for inactivating N-glycosylation sites in proteins include those described in U.S. Patent 5,071,972 and EP 276,846, hereby incorporated by reference.

In another example of variants, sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon folding or renaturation.

Other variants are prepared by modification of adjacent dibasic amino acid residues, to enhance expression in yeast systems in which KEX2 protease activity is present. EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein. KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites.

Encompassed by the invention are oligomers or fusion proteins that contain semaphorin or semaphorin receptor polypeptides. Such oligomers may be in the form of covalently-linked or noncovalently-linked multimers, including dimers, trimers, or higher oligomers. As noted above, preferred polypeptides are soluble and thus these oligomers may comprise soluble polypeptides. In one aspect of the invention, the oligomers maintain the binding ability of the polypeptide components and provide therefor, bivalent, trivalent, etc., binding sites.

i~ WO 01/18044 PCT/US00/24560 One embodiment of the invention is directed to oligomers comprising multiple polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the polypeptides.

Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of the polypeptides attached thereto, as described in more detail below.

As one alternative, an oligomer is prepared using polypeptides derived from immunoglobulins.

Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, by Ashkenazi et al. (PNAS USA 88:10535, 1991); Bym et al. (Nature 344:677, 1990); and Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1 10.19.11, 1992).

One embodiment of the present invention is directed to a dimer comprising two fusion proteins created by fusing a polypeptide of the invention to an Fc polypeptide derived from an antibody. A gene fusion encoding the polypeptide/Fc fusion protein is inserted into an appropriate expression vector. Polypeptide/Fc fusion proteins are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent molecules.

The term "Fc polypeptide" as used herein includes native and mutein forms of polypeptides made up of the Fc region of an antibody comprising any or all of the CH domains of the Fc region.

Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included. Preferred polypeptides comprise an Fc polypeptide derived from a human IgGI antibody.

One suitable Fc polypeptide, described in PCT application WO 93/10151 (hereby incorporated by reference), is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in U.S. Patent 5,457,035 and in Baum et al., (EMBOJ. 13:3992-4001, 1994) incorporated herein by reference. The amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala. The mutein exhibits reduced affinity for Fc receptors.

The above-described fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns.

In other embodiments, the polypeptides of the invention may be substituted for the variable portion of an antibody heavy or light chain. If fusion proteins are made with both heavy and light WO 01/18044 PCT/US00/24560 chains of an antibody, it is possible to form an oligomer with as many as four semaphorin or semaphorin receptor extracellular regions.

Alternatively, the oligomer is a fusion protein comprising multiple polypeptides, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233, which are hereby incorporated by reference. A DNA sequence encoding a desired peptide linker may be inserted between, and in the same reading frame as, the DNA sequences of the invention, using any suitable conventional technique. For example, a chemically synthesized oligonucleotide encoding the linker may be ligated between the sequences. In particular embodiments, a fusion protein comprises from two to four soluble semaphorin or semaphorin receptor polypeptides, separated by peptide linkers.

Another method for preparing the oligomers of the invention involves use of a leucine zipper.

Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Zipper domains (also referred to herein as an oligomerizing, or oligomer-forming, domain) and their use are well-known in the art.

B. ASSAYS The influence of semaphorins on cellular efflux may be used to control the development of MDR phenotypes of a cell or group of cells. For example, semaphorin polypeptides, or polynucleotides encoding semaphorin polypeptides may be administered to a cell or group of cells to stimulate or inhibit cellular efflux, to either induce, enhance, suppress or arrest the development of multiple drug resistance in the target cells. Identification of semaphorin-containing compositions that may be used in this manner may be carried out via a variety of assays known to those skilled in the art.

Included in such assays are those that evaluate the ability of a semaphorin composition to influence cell survival rates in the presence of cytotoxic agents. Such an assay would involve, for example, the determination of sensitivities of tumor cells or cell lines to anticancer drugs in the presence and absence of a semaphorin. In these assays, one would determine a rate of cell death in the presence of the cytotoxic agent (such as doxorubicin, etc.) and then determine if the rate of cell death resulting from that agent is altered in the presence of a semaphorin.

Alternatively, one might monitor MDR protein-like activity in a cell by examining the ability of primary cells, or cells overexpressing MDR proteins, to efflux dyes in the presence and absence of a semaphorin. These types of assays are routine, and employ what are referred to as either "slow" or "fast" cellular dyes, that is, dyes that are typically lipophilic, or cationic. (see, Lelong, et al, 1991). One example of use of these dyes involves loading the dye into a cell at low temperatures, such WO 01/18044 PCT/US00/24560 as 4 degrees (or on ice), and then examining the stained cells by flow cytometry. The cells will fluoresce depending on how much dye they take up; and, if loaded in the presence of an MDR efflux pump inhibitor such as verapamil or cyclosporin, or a semaphorin of the present invention, they may fluoresce more brightly than cells loaded in the absence of an MDR inhibitor. In this manner inhibitors of cellular efflux pumps can be identified. This assay may then also be taken a step further by transferring the cells loaded with dye to elevated temperature conditions, such as 37 degrees Centigrade, for a period of time, such as approximately three hours, at which time the cells are again examined on the flow cytometer, and compared to cells that were loaded with dye and held at cooler temperatures, such as the previously noted 4 degrees. At higher temperatures, cellular efflux pumps, including the MDR proteins, are quite active and can extrude the dye from the cell at a rapid rate. The ability of a semaphorin to influence this efflux can be measured by including the semaphorin in the assay during the efflux phase.

Yet another assay that may be used in the present invention involves examining intracellular pH, and the pH of intracellular compartments, in response to semaphorins. These types of assays again use fluorescent probes that target to the cytoplasm or to specific organelles, and exhibit fluorescence pattern changes as the pH changes. See, e.g. Altan, N et al. J. Exp. Med. 187:1583, Altan, N et al. PNAS 96:4432, Chen, Y et al. JBC 274:18364, Schindler, M. et al. Biochemistry 35:2811.. Dyes that are useful in such assays include dyes such as acridine orange (which targets acidic compartments and whose fluorescent wavelength and intensity depends on the pH of that organelle), BODIPY-ceramide (which targets the trans-golgi network), SNARF-dextrans of varying molecular weights (allowing one to target cytosol or nucleus), and FITC-transferrin or BODIPYtransferrin (which targets endocytic vesicles). These dyes are used to stain cells and then their fluorescence intensity and/or pattern is measured on a confocal microscope.

Another embodiment of the present invention provides a method of detecting the ability of the test compound to influence the MDR phenotype of a cell. In this aspect of the invention, the method includes contacting a first cell with a test compound including a semaphorin or semaphorin receptor in the presence of a cytotoxic agent. The method then involves measuring the rate of death of that first cell. Then the rate of death of a controlled cell is observed, with the control cell under similar conditions but in the absence of a test compound comprising a semaphorin or semaphorin receptor, and in the presence of a cytotoxic agent, which is the same agent administered to the first cell. The death rate of the first cell is then compared to the death rate of the control cell and the difference in the rate of cell death between the first cell and the control cell is indicative of an agent that influences development of multiple drug resistance phenotype. This agent may be one that functions to increase multiple drug resistance in the cell or to decrease multiple drug resistance in the cell.

I" WO 01/18044 PCTIUS00/24560 In addition to those semaphorins listed in Section III above, specific semaphorins that may be tested according to this embodiment of the invention include A39R, DCSema, CD100, Sema III, Sema E, or active fragments of these semaphorins. Specific exemplary useful semaphorin receptors that may be used in these assays include VESPR. Alternatively, an agonist or antagonist to a semaphorin or semaphorin receptor may used according to this aspect of the invention. In a particularly preferred embodiment, an antibody to VESPR is used. Examples of cytotoxic agents that may be used in this method of detection include doxorubicin, radiation, tamoxifen, or any other compound known to have a cytotoxic effect on a cell.

In another aspect, the present invention provides a method of detecting the ability of a test compound to influence the MDR phenotype of a cell by modulating the cellular efflux of that cell. In this aspect, one example of such a method includes: contacting a first cell with a test compound including a semaphorin or semaphorin receptor in the presence of a dye; measuring the net rate of influx of dye into this first cell; and observing the net rate of influx of dye into a control cell under similar conditions, but in the absence of a test compound comprising a semaphorin or semaphorin receptor. In this embodiment, the net rate of influx of dye is the rate of influx of dye relative to the rate of efflux, as measured by the amount of dye detected in the cell. The comparison will provide a difference in the net rate of influx of the dye such that influx of the dye into the first cell relative to the control cell is indicative of an agent that can influence cellular efflux. The test compound may function to either activate or up-regulate, or inhibit or down-regulate cellular efflux, either of which function may be detected through this method.

In addition to the semaphorins listed in Section III above, specific semaphorins that may be tested according to this embodiment of the invention include A39R, DCSema, CD100, Sema III, Sema E, or active fragments of these semaphorins. Specific exemplary useful semaphorin receptors that may be used in these assays include VESPR. Alternatively, an agonist or antagonist to a semaphorin or semaphorin receptor may used according to this aspect of the invention. In a particularly preferred embodiment, an antibody to VESPR is used.

Virtually any dye may be used in this method. Exemplary dyes include those which are characterized by one or more of the following properties: lipophilic, cationic, fluorescent, and radioactive. Specific exemplary dyes include a slow dye; a fast dye; acridine orange; various BODIPY dyes including specific ones such as 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3,5-dipropionic acid, BODIPY ceramide, and BODIPY-transferrin; seminaphthorhodafluors ("SNARF") -dextran; and Fluorescien isothiocyanate ("FITC")-transferrin.

C. COMPOUNDS AND METHODS FOR THE MODULATION OF CELLULAR EFFLUX Described below are methods and compositions employing semaphorins, semaphorin receptors, fragments of these, or the genes encoding them, for use in the promotion or suppression of WO 01/18044 PCTIUSOO/24560 cellular efflux or for controlling development of MDR in a target cell or group of cells. It is specifically contemplated that such compositions and methods can be used to treat a cell or group of cells both in vivo and in vitro.

For example, such methods can comprise administering compounds which modulate cellular efflux, and thereby influence development ofMDR phenotype or cellular efflux-related disease states.

Administration of such compounds can be used to inhibit drug resistance thereby sensitizing cells to cytotoxic substances; to promote resistance to cytotoxic substances and protect against cytotoxic substances; or to the dysregulation of cellular efflux in cells that are unable to otherwise regulate themselves, such as those cells associated with diseases such as cystic fibrosis.

In addition to methods utilizing semaphorin or semaphorin receptor-encoding nucleic acid sequences, it is also useful to modulate cellular efflux by using the semaphorin or semaphorin receptor polypeptide, or polypeptide fragments. Another means of modulating cellular efflux or MDR phenotypes according to the present invention involves the use of any of the compounds identified through the assays set forth in Section B above.

When the actual nucleic acid sequences encoding the semaphorins; semaphorin receptors; or fragments of either that are disclosed in the present invention are delivered according to the methods described herein, it is advantageous to use a delivery mechanism so that the sequences will be incorporated into a cell for expression. Delivery systems that may advantageously be employed in the contemplated methods include the use of, for example, viral delivery systems such as retroviral and adenoviral vectors, as well as non-viral delivery systems. Such delivery systems are well known by those skilled in the art.

In one aspect of the invention, a retroviral delivery system may be employed. The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins.

The integration results in the retention of the viral gene sequences in the recipient cell and its descendants.

The retroviral genome contains three genes gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene, termed i, functions as a signal for packaging of the genome into virions. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990).

In order to construct a retroviral vector, a nucleic acid encoding a Grb2 or Crkl antisense construct is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol and env genes but without the LTR and W components is constructed (Mann et al, 1983). When a recombinant WO 01/18044 PCT/US00/24560 plasmid containing an inserted DNA, together with the retroviral LTR and \y sequences, is introduced into this cell line (by calcium phosphate precipitation for example), the y sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable expression require the division of host cells (Paskind et al., 1975).

Alternatively, an adenoviral delivery system may be employed. Human adenoviruses are double-stranded DNA tumor viruses with genome sizes of approximate 36 kB (Tooze, 1981). As a model system for eukaryotic gene expression, adenoviruses have been widely studied and well characterized, which makes them an attractive system for development ofadenovirus as a gene transfer system. This group of viruses is easy to grow and manipulate, and they exhibit a broad host range in vitro and in vivo.

In lytically infected cells, adenoviruses are capable of shutting off host protein synthesis, directing cellular machineries to synthesize large quantities of viral proteins, and producing copious amounts of virus.

The El region of the genome includes ElA and EIB which encode proteins responsible for transcription regulation of the viral genome, as well as a few cellular genes. E2 expression, including E2A and E2B, allows synthesis of viral replicative functions, e.g. DNA-binding protein, DNA polymerase, and a terminal protein that primes replication. E3 gene products prevent cytolysis by cytotoxic T cells and tumor necrosis factor and appear to be important for viral propagation. Functions associated with the E4 proteins include DNA replication, late gene expression, and host cell shutoff. The late gene products include most of the virion capsid proteins, and these are expressed only after most of the processing of a single primary transcript from the major late promoter has occurred. The major late promoter (MLP) exhibits high efficiency during the late phase of the infection (Stratford-Penricaudet and Perricaudet, 1991).

As only a small portion of the viral genome appears to be required in cis (Tooze, 1981), adenovirus-derived vectors offer excellent potential for the substitution of large DNA fragments when used in connection with cell lines such as 293 cells. AdS-transformed human embryonic kidney cell lines (Graham, et al., 1977) have been developed to provide the essential viral proteins in trans.

Particular advantages of an adenovirus system for delivering foreign proteins to a cell include (i) the ability to substitute relatively large pieces of viral DNA by foreign DNA; (ii) the structural stability of recombinant adenoviruses; (iii) the safety of adenoviral administration to humans; and (iv) lack of any known association of adenoviral infection with cancer or malignancies; the ability to obtain high titers of the recombinant virus; and (vi) the high infectivity of adenovirus.

Further advantages of adenovirus vectors over retroviruses include the higher levels of gene expression. Additionally, adenovirus replication is independent of host gene replication, unlike retroviral WO 01/18044 PCT/US00/24560 sequences. Because adenovirus transforming genes in the El region can be readily deleted and still provide efficient expression vectors, oncogenic risk from adenovirus vectors is thought to be negligible (Grunhaus Horwitz, 1992).

In general, adenovirus gene transfer systems are based upon recombinant, engineered adenovirus which is rendered replication-incompetent by deletion of a portion of its genome, such as El, and yet still retains its competency for infection. Sequences encoding relatively large foreign proteins can be expressed when additional deletions are made in the adenovirus genome. For example, adenoviruses deleted in both El and E3 regions are capable of carrying up to 10 kB of foreign DNA and can be grown to high titers in 293 cells (Stratford-Perricaudet and Perricaudet, 1991). Surprisingly persistent expression of transgenes following adenoviral infection has also been reported.

Other viral vectors may be employed as expression constructs in the present invention. Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988) adeno-associated virus (AAV) (Ridgeway, 1988; Baichwal and Sugden, 1986; Hermonat and Muzycska, 1984) and herpesviruses may be employed. They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).

With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. The hepatotropism and persistence (integration) were particularly attractive properties for liver-directed gene transfer. Chang et al. recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al., 1991).

In yet another aspect, non-viral vectors may be used according to the presently disclosed methods.

Several non-viral methods for the transfer of expression vectors into cultured mammalian cells also are contemplated by the present invention. These include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al., 1986; Potter et 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al., 1987), gene bombardment using high velocity microprojectiles (Yang et al., 1990), polycations (Boussifet al., 1995) and receptor-mediated transfection WO 01/18044 PCTIUS00/24560 (Wu and Wu, 1987; Wu and Wu, 1988). Some of these techniques may be successfully adapted for in vivo or ex vivo use.

In one embodiment of the invention, the expression construct may simply consist of naked recombinant vector. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. For example, Dubensky et a. (1984) successfully injected polyomavirus DNA in the form of CaPO 4 precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of CaPO 4 precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding an Grb2 or Crkl construct may also be transferred in a similar manner in vivo.

Another embodiment of the invention for transferring a naked DNA expression vector into cells may involve particle bombardment. This method depends on the ability to accelerate DNA coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987). Several devices for accelerating small particles have been developed. One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al., 1990). The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads.

Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et 1990; Zelenin et 1991). This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ. DNA encoding a Grb2 or Crkl construct may be delivered via this method.

Alternatively, the degree of cellular efflux in a cell may be influenced by administering a compound identified via one of the assays described above, that increases or decreases the rate of cellular efflux or development ofMDR phenotype.

D. FORMULATION AND ADMINISTRATION OF THE DISCLOSED COMPOSITIONS The formulations described herein may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose or polyoxyethylenesorbitans. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride as described above. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate or gelatin. Other agents that may be employed include, but are not limited to lecithin, urea, ethylene oxide, propylene oxide, hydroxypropylcellulose, methylcellulose, or polyethylene glycol.

Aqueous compositions (inocula) as described herein may include an effective amount of a desired pharmacologically active agent dissolved or dispersed in a pharmaceutically acceptable aqueous medium. Such compositions are also referred to as inocula. The use of pharmaceutically I' WO 01/18044 PCT/US00/24560 acceptable carrier media and agents for pharmaceutically active substances is well known in the art.

Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions as described above.

A semaphorin used in the present invention may be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and those that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.

Such compositions of the present invention can be, alternatively, complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc. or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or sphereoblasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance, and are thus chosen according to the intended application.

The therapeutic compositions of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection may also be prepared. Alternatively, the compositions of the present invention may be administered as inhalants in an aerosolized form. Depending upon the needs of the formulator, administrator, or the subject of the treatment, the presently disclosed compositions may take virtually any form including liquid, suspension, aerosol, emulsion, solution, oil, mixture, cream, ointment, gel, suppository, semi-solid, aerosol, powder, lyophilized form that may be reconstituted when appropriate, tablet, capsule or any other form or state convenient for administration to treat the described disorders. A typical composition comprises a pharmaceutically acceptable carrier.

The presently disclosed compositions and methods may utilize both oral and non-oral administration routes to influence the target cell or cells including, for example, by injection via the intradermal, subcutaneous, and intravenous routes; by transdermal delivery; by inhalation or buccal delivery, or by ingestion of tablets or capsules. For example, local or regional delivery of compounds to a cell or cells can be by injection into the tissue, injection into the vasculature or lymphatics to effect regional infusion, inhalation, or regional perfusion by use of an extracorporeal circuit.

Administration in a targeted fashion is useful to, for example, more effectively eliminate neoplastic cells, while minimizing the adverse effects of chemotherapy on healthy cells. For example, an inhibitor of cellular efflux can be directly administered, according to the methods disclosed herein, to WO 01/18044 PCT/US00/24560 neoplastic cells such as tumor cells, to prevent their development of MDR and thereby promote their susceptibility to chemotherapeutic or otherwise cytotoxic agents, while simultaneously administering to healthy cells a promoter of cellular-efflux to prevent their destruction by cytotoxic agents.

The optimal daily dose of semaphorin, semaphorin receptor such as VESPR or soluble VESPR, or of an agonist or antagonist of one of these, alone or in combination, useful for the purposes of the present invention is determined by methods known in the art. For example, dosages can be determined based on the severity of the disease or condition being treated, the condition of the subject to whom treatment is being given, the desired degree of therapeutic response, and any concomitant therapies being administered to the subject. Ordinarily, however, administration will be such that a serum level of between about 100ng/ml to about 100.g/ml of semaphorin, semaphorin receptor, or agonist or antagonist of either, is achieved. Preferred doses will achieve blood serum levels of between 500ng/ml and 1g/ml. The dose can be administered in a single or multiple dosage regimen, or may be by a method that allows for a continuous release of relatively small amounts of the active ingredient from a single dosage unit, such as by a transdermal patch or ingested extended release capsule, over the course of one or more days.

To determine when inhibition or retardation of the various target diseases or conditions, or when amelioration, regression or destruction of the targeted diseases or conditions has been achieved, any of the following can be considered: improvement in patient condition or quality of life; increased longevity of life; decreased pain; decreased severity of symptoms of the targeted disease or condition; retardation of abnormal tissue growth or metastases such as in the case of suppression of development of MDR in cells being targeted for cancer chemotherapeutic disease; an increase in desired tissue growth or viability in the case or promotion of drug resistance in healthy tissue and cells; and the like.

Any of these endpoints as well as others may be considered to determine the effectiveness of the therapy, and may be measured or determined by patient self-evaluation; objective screening; or by diagnostic testing such as by X-ray, CT or PET scanning or the like.

The compositions as described herein may be formulated so that they are contained in a vial, bottle, tube, syringe inhaler or other container for single or multiple administrations. Such containers may be made of glass or a polymer material such as polypropylene, polyethylene, or polyvinylchloride, for example. Preferred containers may include a seal, or other closure system, such as a rubber stopper that may be penetrated by a needle in order to withdraw a single dose and then reseal upon removal of the needle. All such containers for injectable liquids, lyophilized formulations, reconstituted lyophilized formulations or reconstitutable powders for injection known in the art or for the administration of aerosolized compositions are contemplated for use in the presently disclosed compositions and methods.

WO 01/18044 PCT/US00/24560 In alternative embodiments, the presently disclosed compositions are administered in conjunction, either simultaneously or sequentially, with additional active agents such as an immunosuppressant, cell sensitizer, or other chemotherapeutic agent including a cancer chemotherapeutic agent. Exemplary agents to be used in combination with the presently disclosed compositions include cyclosporin, tamoxifen, FK506, taxotere, doxorubicin, cis-platin, I-phosphamide, or methotrexate.

WO 01/18044 PCTIUSOO/24560

REFERENCES

Altan, et al., J. Exp. Med. 187:1583 Altan, et al., PNAS 96:4432 Chen, et al., JBC 274:18364 Kim et al., Blood, 91:4106-4117 (1998) Lelong, et Molecular Pharmacology 40:490 (1991).

Schindler, el al., Biochemistry 35:2811 Weisburg et al, J. Biol Chem, 274, 10877-88 (1999) Yamada et.aI., PNAS 94:14713-14718 (1997) Zaman, G.J.R. eta!., PNAS USA 91:8822 (1994)

Claims (14)

1. A method of modulating cellular efflux comprising contacting a cell with an agent selected from the group consisting of: a semophorin polypeptide; a semaphorin receptor polypeptide; a polynucleotide encoding a semaphorin polypeptide; a polynucleotide encoding a semaphorin receptor polypeptide; an agonist of a semaphorin polypeptide or semaphorin receptor polypeptide; and an antagonist of a semaphorin polypeptide or semaphorin receptor polypeptide; wherein contact with said agent is effective to activate or inhibit cellular efflux in said cell.

2. The method of claim 1, wherein said semaphorin polypeptide is A39R, DCSema, 15 CD100, Sema II or Sema E.

3. The method of claim 1, wherein said semaphorin receptor polypeptide comprises the amino acid sequence of SEQ ID NO: 2.

4. The method of claim 1, wherein said polynucleotide encoding said semaphorin receptor polypeptide comprises the nucleic acid sequence of SEQ ID NO: 1.

5. The method of claim 1, wherein said agonist or antagonist is an antibody, an antigen or a small molecule. o

6. The method of claim 5, wherein said antibody is an antibody to VESPR.

7. The method of any preceding claim, wherein said agent is administered in conjunction with a cytotoxic or sensitizing agent. 25 8. The method of claim 7, wherein said cytotoxic or sensitizing agent is tamoxifen, cisplatin, doxorubicin, radiation, methotrexate, cyclosporin, taxotere, FK506 or i- phosphamide.

9. A method of influencing multiple drug resistance in a cell, comprising contacting said cell with an agent selected from the group consisting of: a semaphorin polypeptide; a semaphorin receptor polypeptide; i- a polynucleotide encoding a semaphorin polypeptide; a polynucleotide encoding a semaphorin receptor polypeptide; an agonist of a semaphorin polypeptide or semaphorin receptor polypeptide; and an antagonist of a semaphorin polypeptide or semaphorin receptor polypeptide; wherein contact with said agent is effective to promote or suppress development of multiple drug resistance in said cell. A method ofdetecting the ability ofa test compound to effect the MDR phenotype of a cell, said method comprising: contacting a first cell with a test compound and either a semaphorin or semaphorin receptor, in the presence of a cytotoxic or sensitising agent; measuring the rate of death of said first cell; S- observing the rate of death of a control cell under similar conditions but in 15 the absence of said test compound; comparing said rate of death of said first cell to the rate of death of said control cell; wherein a difference in the rate of death of said first cell relative to said control cell is indicative of an effector of MDR phenotype.

11. The method of claim 10, wherein said cytotoxic or sensitising agent is tamoxifen, cisplatin, doxorubicin, radiation, methotrexate, cyclosporin, taxotere, FK506 or i- V phosphamide.

12. The method of claim 10 or claim 11, wherein said test compound is A39R, DCSema, CD100, Sema I, or Sema E. or an antibody to VESPR. 25 13. A method of detecting the ability of a test compound to effect the MDR phenotype ofa cell by modulating cellular efflux in said cell, said method comprising: contacting a first cell with a test compound and either a semaphorin or semaphorin receptor, said first cell in the presence of a dye; measuring the net rate of influx of dye into said first cell; observing the net rate of influx of dye into a control cell under similar conditions, but in the absence of said test compound; and comparing said net rate influx of dye into said first cell to said net rate of influx of dye into said control cell; wherein a difference in the net rate of influx of dye into said first cell relative to said control cell is indicative of an effector of cellular efflux.

14. The method of claim 13, wherein said test compound is A39R, DCSema, CD100, Sema II, or Sema E. or an antibody to VESPR. The method of claim 13 or claim 14, wherein said dye is characterised by one or more of the following properties: lipophilic, cationic, fluorescent and radioactive.

16. The method of claim 13 or claim 14, wherein said dye is a slow dye, a fast dye, acridine orange, BODIPY ceramide, SNARF-dextran, FITC-transferrin or BODIPY- transferrin.

17. Use of an agent selected from the group consisting of: a semaphorin polypeptide; a semaphorin receptor polypeptide; 15 a polynucleotide encoding a semaphorin polynucleotide; a polynucleotide encoding a semaphorin receptor polynucleotide; an agonist of a semaphorin polypeptide or semaphorin receptor polypeptide; and an antagonist of a semaphorin polypeptide or semaphorin receptor polypeptide; in the manufacture of a medicament for the treatment of a cellular-efflux related disease or condition.

18. Use according to claim 17, wherein the medicament is for said treatment in conjunction with a cytotoxic or sensitising agent. 25 19. Use according to claim 18, wherein said cytotoxic or sensitising agent is tamoxifen, cisplatin, doxorubicin, radiation, methotrexate, cyclosporin, taxotere, FK506 or i- phosphamide. Use according to any of claims 17 to 19, wherein said cellular-efflux-related disease or condition is multiple drug resistance, a neoplastic disease, cystic fibrosis or an immunodeficiency disorder. DATED: 24 March, 2003 PHILLIPS ORMONDE FITZPATRICK Attorneys for: IMMUNEX CORPORATION WO 01/18044 PCTUSOO/24560 INFORM4ATION FOR SEQ ID NO:l: (i)SEQUENCE CHARACTERISTICS: LENGTH: 4707 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (ix) FEATURE: NAME/KEY: CDS LOCATION: 1. .4707 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: ATG GAG GTC TCC CGG AGG AAG GCG CCG CCG CGC CCC CCG CGC CCC GCA Met Glu Val Ser Arg Arg Lys Ala Pro Pro Arg Pro Pro Arg Pro Ala I GCG Ala CGG Arg ATC Ile CTG Leu CGG Arg GCG Ala CGC Arg CCA Pro GCC Cly C Ala GAC Asp GAC Asp CGG Arg GAG Glu CTG Leu CCG Ala CCG AGC Ala Ser CAG CTG Cln Leu CAA GCG Gin Ala CCC CGG Pro Arg 100 COG CCG Gly Ala 115 5 CTG Leu GAG Glu CAG Cln GAC Asp GCC Gly CCC Pro GCC Ala GCC TAT Ala Tyr CTC TGC Val Trp, 40 GAC GGC Asp Cly 55 AGC CTG Ser Leu TGC ACA Cys Thr AGC AC Ser Ser CTC GGG Leu Gly 120 10 CTG CTG Leu Leu CGG TCG Arg Ser GTG TTT Val Phe GAG CAC Glu His GAG CCC, Clu Pro 90 TTC AGC Phe Ser 105 GO CTC Gly Leu GCA CTG CC Ala Leu Ala GAG CAA GCC Clu Gin Ala GTG C AGC Val Ala Ser ACC CTC TCC Ser Leu Ser 75 GTC TCG CTG Val Ser Leu AAG CTG CTG Lys Leu Leu OTG CTC ACC Leu Leu Thr 125 OCT Ala ATC Ile GGC Giy CGC Arg GCG Ala CTG Leu 110 GC Gly CCC Pro GGA Gly AC Ser CTC Leu CCC Pro CCC Pro TGG Trp GCC Gly GCC Ala TOC Cys TAC Tyr CCC Pro TAC Tyr ACC Thr 96 144 192 240 *288 336 384 TTC GAC CCC GGC GCC TGC GAG GTG CG CGCCAC T G CTG GGC AAC CTG AGC CGC WO 01/18044 WO 01/ 8044PCTUSOO/24560 Phe As 13 AAC TC Asn Se 145 TCG AC Ser Th: CTG GO Leu Al~ CGC TGC Arg CyE GAC ACC Asp Thr 210 CTG TGC Leu Cys 225 AAC GGC Asn Gly GCT 0CC Ala Ala GTG CTG Val Leu GAC GGC Asp Gly 290 GTC TG Val Trp 305 CGC TCC Arg Ser CAG GCG Gin Ala p Arg Gly Ala C3 0 C CTG CGC AAC GC r Leu Arg Asn G] G GCC GGC GTO G r Ala Gly Val Va 165 3GTG 0CC 0CC AC a Val Ala Ala Th 180 AAC CCC GCG C Asn Pro Ala Al. 195 GAG GGG CGC AGi Glu Gly Arg Se: GAG GOC GCG GG( Oiu Gly Ala Gil 23( AGC ATC TAC TT( Ser Ile Tyr Phe 245 ACC GGC TGG CCC Thr Gly Trp Pro 260 TTC CAG GGC CAG Phe Gin Gly Girt 275 CGC CGC CTG CTC Arg Arg Leu Leu GCG GGA GTG TTC Ala Gly Val Phe 310 CCC ACC ACC ACO Pro Thr Thr Thr 325 CGC GCC AAG AGG Arg Ala Lys Arg 340 Is 0 Giu V~ 135 ACC G Thr G] 'G TAC 1 Tyr C TAC r Tyr A~ TCC a Ser CCTG r Leu 215 AGC rSer CCC Pro AGC Ser OCA T1 Ala S 2 CTC TI Leu S 295 AOC G Ser A GCG C' Ala L GTC A( Val Si CG Ax 07 Va GA As 20 GC' Al Lje TAC ~TC ~et ~cc :er :80 'cc er CG la TC C al1 Arg Pi kG GTO G7 Lu Val Va C GCG GO *g Ala 01 17 'G CTG CC, 1 Leu Pri 185 C CAC GA( p His Asi 0 C ACG CAC a Thr Gir 3CAC TTC a His Phe TAC CCC Tyr Pro 250 OCG COC *Ala Arg 265 CTC GAC *Leu Asp TCC AGC Ser Ser 0CC'OCT Ala Ala TGC CTC Cys Leu 330 TOO GAC Trp Asp 345 -0 Leu Gly 140 'G TCG TGC I Ser Cys 155 C COO AAC y Arg Asn 0 r GAG CCG 3 Glu Pro ACG 0CC. SThr Ala GAG CTG i Glu Leu 220 GTG GAC Val Asp2 235 TAC AAC Tyr Asn ATC GCC Ile Ala G TOC GGCC Cys Gly H 2 CTA GTG 0 Leu Val G 300 GOA GAG G Gly Giu G: 315 TTC AGA AX Phe Arg m( TTC AAG A( Phe Lys Th A H: Al As AT 1l 20 30 31 'AC ln AC is AG lu Ly sn Leu .C CCG as Pro ~C CGC *n Arg LACO .u Thr 190 'C GCG e Ala G CGC y Arg CTTT a Phe I [ACO 7Thr S 2 AGC SerT 270 GGC C Gly H cc C' Ala Li CAG 0 Gin GI AGT G Ser G01 33 GCC GA Ala Gi1 350 S e G01 TG Tr 17 Al CTc Let CTC !TC jeu LGC e r .cc hr AC TG eu Lu iS U ~r Arg ~GGC n Gly 160 G TAC pTyr G AGC a Ser C AAG .I Lys AAG ILys TOG ITrp 240 *0C Gly GAG Giu CCC Pro GAC Asp CG Arg 320 ATC Ile AGC Ser 480 528 576 624 672 720 768 816 864 912 960 1008 1056 WO 01/18044 WO 0118044PCT/USOO/24560 CAC TGC AAA GAA GGG GAT CAA CCT GAA AGA GTC CAA CCA ATC GCA TCA His Cys Lys 355 Giu Gly Asp Gin Giu Arg Val Gin Ile Ala Ser 1104 1152 TCT ACC Scr Thr 370 TTG ATC CAT TCC Leu Ile His Ser CTG ACA TCC GTT TAT GGC ACC GTG GTA Leu Thr Ser Val Tyr Gly Thr Val Vai 380 AAC AGG ACT GTT Asn Arg Thr Val TTA TTC Leu Phe 390 TTG GGG ACT Leu Gly Thr OGA GAT GGC CAG Gly Asp Giy Gin 395 AAT TGT CCA GAG Asn Cys Pro Giu TTA CTT Leu Leu 400 OTT ATC Val Ile 415 AAG GTT ATT CT Lys Vai Ile Leu GAG AAT TTG ACT Giu Asn Leu Thr TAT GAA ATT Tyr Giu Ile CCT OTG AAG Pro Vai Lys 435 AAA Lys 420 GAA GAG ACA CCT Glu Glu Thr Pro TTC TAC AAA CTC Phe Tyr Lys Leu GTT CCT GAT Val Pro Asp 430 GAG GTG AGG Giu Val Arg AAT ATC TAC ATT Asn Ile Tyr Ile CTA ACA OCT 000 Leu Thr Ala Gly 1200 1248 1296 1344 1392 1440 1488 AGA ATT Arg Ile 450 CGT GTT GCA AAC Arg Val Ala Asn AAT AAA CAT AAA Asn Lys His Lys TCC Ser 460 TOT TCG GAG TGT Cys Ser Glu Cys ACA GCC ACA GAC Thr Ala Thr Asp CAC TOC GOT TOO His Cys Gly Trp CAT TCO CTA CAA His Ser Leu Gin TOC ACT TTT CAA Cys Thr Phe Gin GAT TGT OTA CAT Asp Cys Val His GAG AAC TTA GAA Oiu Asn Leu Glu AAC TOO Asn Trp 495 CTG OAT ATT Leu Asp Ile COA AGC AGT Arg Ser Ser TCT OGA GCA AAA Ser Gly Ala Lys TOC CCT AAA ATT Cys Pro Lys Ile CAG ATA ATT Gin Ile Ile 510 AGC TTC TCT Ser Phe Ser 1536 1584 AAA GAA AAG ACT Lys Giu Lys Thr ACA Thr 520 GTG ACT ATG GTG Val Thr Met Val CCA AGA Pro Arg 530 CTC TGC Leu Cys 545 CAC TCA AAG TOC His Ser Lys Cys OTO AAG ANT OTO GAC TCT AGC AOG GAG Val Lys Asn Val Asp Ser Ser Arg Giu 1632 1680 CAG AAT AAA AGT CAG CCC AAC CG Gin Asn Lys Ser Gin Pro Asn Arg 550 ACC TGC Thr Cys 555 ACC TOT AGC Thr Cys Ser CCA ACC AGA GCA ACC TAC Pro Thr Arg Ala Thr Tyr 565 AAA OAT OTT TCA Lys Asp Val Ser 570 OTT OTC AAC OTG ATG TTC Val Val Asn Val Met Phe 575 1728 WO 01/18044 Wa 0118044PCT/USOO/24560 TCC TTC GG' Ser Phe G11 TCA TCA TTI Ser Ser Let 59! TGT AAA AG'] Cys Lys Sei 610 TCT GAT TAI1 Scr Asp Tyr 625 ACA TCA GGA Thr Ser Gly CAG GCT TTA Gin Ala Leu TCG ACA TTA Ser Thr Leu 675 CGG GCA TCG Arg Ala Ser 690 AAG GAT GTG Lys Asp Val 705 TTC TCT CTT Phe Ser Leu TTT GAT GGT Phe Asp Gly CTG CCA CAT Leu Pro His 755 GGT CAA AAT Gly Gin Asn 770 V' TCT TG( ~Ser Txj 580 SAAA GA7 1 Lys Git GCA AG; *Ala Arc *GAG AGAi *Giu Arg GGA GGA Gly Gly 645 CAG GTC Gin Val 660 GGG AAA Gly Lys AAC ATC Asn Ile ATA CAG Ile Gin CCA TCA Pro Ser 725 GGG AAC Cly Asn 740 TGT TCC Cys Scr ATA ACC Ile Thr AAT TTA TCA SAsn Leu 5cr tTGC CCA GCA 1Cys Pro Ala 600 AGG TGT ATC Arg Cys Ile 615 AAC CAG GAA Asn Gin Giu 630 AGA CCC AAG Arg Pro Lys TTC TAC ATT Phe Tyr Ile AGC AAC GTG Ser Asn Val 680 ACA ATG ATC Thr Met Ile 695 GTT AGC CAT Val Ser His 710 ACC CGG AAA Ser Arg Lys TGC TCT TCT Cys Ser Ser CTT ATA T'T Leu Ile Phe 1 760 ATG ATG GGC Met Met Cly 775 GAC AGA TTC Asp Arg Phe 585 TGC GTA CAA Cys Vai Ciu CAC CCC TTC AAC TTT Asn Phe ACT GC Thr Cly 605 ACA GCT AAC TGC Asn Cys GCG TGG Aia Trp 1776 1824 1872 1920 His CAG Gin GAG Giu AAG Lys 665 ATA Ile CTG Leu GTG Vl GAA .lu 'TG lai ~CT ~ro, LGA Lrg Pro Phe Thr 620 TGT CCA GTG Cys Pro Vai 635 AAC AAG CG Asn Lys Gly 650 TCC ATT GAG Ser Ile Ciu GTA ACG GGA Val Thr Gly AAA CGA ACC Lys Giy Thr 700 CTA AAT GAC. Leu Asn Asp 715 ATG AAG GAT Met Lys Asp 730 GGA TCC TTA Cly Ser Leu GCT ACC ACC Aia Thr Thr Ala Cys Asp Pro GCT Ala AAC Asn CCA Pro GCA Ala 685 AGT Ser ACC Thr. GTG GTC GAG AAG Val GlU Lys AGA Arg CAC Gin 670 AAC Asn ACC Thr CAC His TGT ACC Thr 655 AAA Lys TTT Phe TGT Cys ATG Met ATC AAC Asn GTA Val ACC Thr GAT Asp AAA Lys 720 CAG Gin 1968 2016 2064 2112 2160 2208 2256 2304 2352 2400 Val Cys Ile rCC TAC ATT GCT AAT TTT CAT GTA ATT GAC AAC Asn Phe Asp Val Ile Asp Asn 780 TTA ATC ATT TCA CAT GAA TTA AAA GCA AAC ATA AAT CTC TCT GAA TAT Leu Ile Ile Sen His Giu Leu Lys Giy Asn 4 Ile Asn Val Ser Giu Tyr W001/18044 WO 0118044PCTTJSOO/24560 795 000 TTT TTA 0CC CCC AGT TTA AAG AGTI GTG GCG ACT TAC Cys Val Ala AAA GTG CGC Lys Val Arg TTG CAT TGT Leu Asp Cys 835 TAT CGG OTO Tyr Arg Val 850 AAIA GAA AAT L~ys Glu Asn 865 CTC TTC CAT Leu Phe His ACT AGA AAT Thr Arg Asn AAG ACT GCA Lys Thr Ala 915 CTG OGA AAC Leu Gly Asn 930 TGG TAT TTT Trp Tyr Phe 945 GCG GCC GTG Ala Ala Val CAG AGT CAA Gin Ser Gin COT GAC GGC Arg Asp Gly Thr Tyr 805 ACG AAT Thr Asn 820 GGA ACC Gly Thr GAA TCC Glu Ser GAC AAC Asp Asn GGC GAA Gly Glu 885 CAA OAT Gin Asp 900 AGC ACC Ser Thr CTG GAG Leu Giu CTG ATT Leu Ile GGG GTG Giy Val 965 AA CTA fin Leu 98 0 rITT OCT ?he AlaC Cys Oly Phe Leu GTC ACT Val Thr CTG Leu GAG Oiu TTC Phe 870 AAT Asn CTT Leu ATT Ile CTC Leu GTG Val1 950 A~CC rhr 3AA 3lu .AG flu CAG Gin OTG Val 855 AAT Asn GG Gly ACC Thr 0CC Ala TAC Tyr 935 CTC Leu AGO Arg TTG Leu CTG Leu OTG AAG Val Lys 825 TAT COO Tyr Arg 840 OAC ACA Asp Thr ATT TCC Ile Ser CAA TTA Gin Leu ACC ATC Thr Ile 905 AAC TCT Asn Ser 920 GTC GAG Vai Oiu CCT OTC Pro Val CAC AAA His Lys CTG GAA Leu Oiu 985 CAG ATG Gin Met Ala 810 CTG Leu GAG Olu GAA Giu AAA Lys AAT Asn 890 CTT Leu TCT Ser CAG Gin TTO Leu TCG Ser 970 AGC Ser GAT Asp *Pro Ser AGA GTA Arg Val GAC CCC Asp Pro CTG GAA Leu Giu 860 AAA GAC Lys Asp 875 TGC ACT Cys Ser TOC AAA Cys Lys AAG AAA Lys Lys GAO TCA Oiu Ser 940 CTA OG Leu Val 955 AAO GAO Lys Giu GAG CTC Oiu Leu AAA TTO Lys Leu Leu CAA Gin AGA Arg 845 GTG Val ATT Ile TTT Phe ATT Ile GTT Val 925 GTT Val PITT Ile CTG Leu 'AT ksp Lys GAC Asp 830 TTC Phe AAA Lys GAA Glu GAA Oiu AAA Lys 910 CCC Arg CCT Pro GCC Val ACT Ser AAA4 Lys 990 OTO Val Ser 815 ACC Thr ACG Tbr ATT Ile ATT Ile AAT Asn 895 GCC Gly GTC Val TCC Ser ATT le CC 975 GAG Glu GTT V~al 800 TCA Ser TAC Tyr 000 Giy CAA Gin ACT Thr 880 ATT le ATC Ile AAG Lys ACA Thr TTT Phe 960 AAA Lys ATA Ile GAT Asp 2448 2496 2544 2592 2640 2688 2736 2784 2832 2880 2928 2976 3024 995 1000 1005 AGT TTT OGA ACT OTT CCC TTC CTT GAC AACTTTOTCGAA 37 AAA CAT TTT GCT CTG AGA 3072 WO 01/18044 wo 01/ 8044PCTIUSOO/24560 Ser Phe Gly Thr Val Pro Phe Leu Asp Tyr Lys His Phe Ala Leu Arg 1010 1015 1020 ACT TTC TTC CCT GAG TCA GGT GGC TTC ACC CAC ATC TTC ACT GAA GAT 3120 Thr Phe Phe Pro Giu Ser Gly Gly Phe Thr His Ile Phe Thr Giu Asp 1025 1030 1035 1040 ATG CAT AAC AGA GAC GCC AAC GAC AAG AAT GAA AGT OTC ACA GCT TTG 3168 Met His Asn Arg Asp Ala Asn Asp Lys Asn Glu Ser Leu Thr Ala Leu 1045 1050 1055 GAT GCC CTA ATC TGT AAT AAA AGC TTT OTT GTT ACT GTC ATC CAC ACC 3216 Asp Ala Leu Ile Cys Asn Lys Ser Phe Leu Val Thr Val Ile His Thr 1060 1065 1070 CTT GAA AAG CAG AAG AAC TTT TCT GTG AAG GAC AGG TGT CTG TTT 0CC 3264 Leu Glu Lys Gin Lys Asn Phe Set Val Lys Asp Axg Cys Leu Phe Ala 1075 1080 1085 TCC TTC OTA ACC ATT CCA CTG CAA ACC AAG CTG GTC TAO CTG ACC AGO 3312 Ser Phe Leu Thr Ile Ala Leu Gin Thr Lys Leu Val Tyr Leu Thr Ser 1090 1095 1100 ATC OTA GAG GTG CTG ACC AGO GAO TTG ATG GAP. CAG TGT AGT AAO ATG 3360 Ile Leu Glu Val Leu Thr Arg Asp Leu Met Glu Gin Cys Ser Asn Met 1105 1110 1115 1120 CAG COG AAA CTO ATG CTG AGA OGO ACG GAG TCC GTC GTC GAA AAA CTC 3408 Gin Pro Lys Leu Met Leu Arg Arg Thr Giu Ser Val Val Glu Lys Leu 1125 1130 1135 OTC ACA AAO TOG ATG TCC OTO TOO CTT TOT OGA TTT OTC OGO GAG ACT 3456 Leu Thr Asn Trp, Met Ser Val Cys Leu Ser Gly Phe Leu Arg Giu Thr 1140 1145 1150 GTO GGA GAG CCC TTC TAT TTG CTG GTG ACO ACT CTG AAO CAG AAA ATT 3504 Val Gly Giu Pro Phe Tyr Leu Leu Val Thr Thr Leu Asn Gin Lys Ile 1155 1160 1165 AAC AAG GOT COO GTG GAT GTA ATO ACT TGC AAA GCC OTO TAC ACA OTT 3552 Asn Lys Gly Pro Val Asp Val Ile Thr Cys Lys Ala Leu Tyr Thr Leu 1170 1175 1180 AAT GAA GAO TGG CTG TTG TGG CAG GTT COG GAA TTO AGT ACT GTG GOA 3600 Asn Oiu Asp Trp Leu Leu Trp Gin Vai Pro Giu Phe Set Thr Val Ala 1185 1190 1195 1200 TTA AAO GTO GTC TTT GAA AAA ATO CCG GAA AAC GAG AGT GCA GAT OTC 3648 Leu Asn Val Val Phe Giu Lys Ile Pro Giu Asn Glu Ser Ala Asp Val 1205 1210 1215 TOT COG AAT ATT TCA GTO AAT GTT CTO GAO TOT GAO ACC ATT GO CAA 3696 Cys Arg Asn Ile Ser Val Asn Val Leu Asp Cys Asp Thr Ile Oly Gin 1220 1225 1230 WO 01/18044 PTUO/46 PCTIUSOO/24560 0CC AAA GAA BAG ATT TTC CAA GCA TTC TTA AGC AAA AAT GOC TCT CCT 3744 Ala Lys Olu Lys Ile Phe Gin Ala Phe Leu Ser Lys Asn Gly Ser Pro 1235 1240 1245 TAT GGA CTT CAG CIT BAT GAA ATT GGT CTT GAG CTT CAA ATG GGC ACA 3792 Tyr Gly Leu Gin Leu Asn Glu Ile Gly Leu Giu Leu Gin Met Gly Thr 1250 1255 1260 CGA CAG AAA OAA CTT CTG GAC ATC GAC AGT TCC TCC GTG ATT CTT GAA 3840 Arg Gin Lys Oiu Leu Leu Asp Ile Asp Ser Ser Ser Val Ile Leu Giu 1265 1270 1275 1280 GAT OGA ATC ACC BAG CTA ABC ACC ATT GGC CAC TAT GAG ATA ICA BAT 3888 Asp Oly Ile Thr Lys Leu Asn Thr Ile Gly His Tyr Glu Ile Ser Asn 1285 1290 1295 GGA TCC ACT ATA AAA GTC ITT BAG BAG ATA OCA BAT ITT ACT ICA OAT 3936 Gly Ser Thr Ile Lys Val Phe Lys Lys Ile Ala Asn Phe Thr Ser Asp 1300 1305 1310 GTG GAG TAC TCO OAT GAC CAC TOC CAT ITO AlT TTA CCA OAT TCO OBA 3984 Val Giu Tyr Ser Asp Asp His Cys His Leu Ile Leu Pro Asp Ser Giu 1315 1320 1325 GCA TTC CAA OAT GIG CBA OGA BAG AGA CAT CGA 000 BAG CAC BAG TTC 4032 Aia Phe Gin Asp Val Gin Gly Lys Arg His Arg Gly Lys His Lys Phe 1330 1335 1340 AAA GTA ABA OBA ATG TAT CTG ACA BAG CTG CTG TCG ACC BAG, OTG OCA 4080 Lys Vai Lys Giu Met Tyr Leu Thr Lys Leu Leu Ser Thr Lys Val Ala 1345 1350 1355 1360 ATT CAT TCT GTG CrT GBA BAA CIT ITT AGA AGO ATT TOO AGl TIA CCC 4128 Ile His Ser Val Leu Glu Lys Leu Phe Arg Ser Ile Trp Ser Leu Pro 1365 1370 1375 AAC AUC AGA OCT CCA TTT OCT AlA AAA TAC TTT TTT GAC TTT ITO GAO 4176 Asn Ser Brg Ala Pro Phe Ala Ile Lys Tyr Phe Phe Asp Phe Leu Asp 1380 1385 1390 GCC CAG OCT OBA BAC AAA AAA ATO ACA OAT CCT GAO 010 GTA CAT AlT 4224 Ala Gin Ala Glu Asn Lys Lys Ile Ihr Asp Pro Asp Vai Val His Ile 1395 1400 1405 TOO BAA ACA BAC AGO CIT CCI OTT CGC TIC TOG OTA BAC ATC 010 BAG 4272 Irp Lys Thr Asn Ser Leu Pro Leu Brg Phe Trp Val Asn Ile Leu Lys 1410 1415 1420 AAC CCT CAG ITT GIC TTT GAO ATT BAG BAG ACA OCA CAT ATA GAO GOC 4320 Asn Pro Gin Phe Val Phe Asp Ile Lys Lys Thr Pro His Ile Asp Gly 1425 1430 1435 1440 TOT ITO ICA OTO AlT 0CC CAG OCA TTC ATO OAT OCA ITT TOT CTC ACA 4368 Cys Leu Ser Val Ile Ala Gin Ala Phe Met Asp Ala Phe Ser Leu Ihr 1445 1450 1455 WO 01/18044 WO 0118044PCTIUSOOI24560 GAG CAG CAA CTA GGG Giu Gin Gin Leu Gly 1460 AAG GAA GCA CCA ACT AAT AAG Lys Giu Ala Pro Thr Asn Lys 1465 OTT CTC TAT GCC Leu Leu Tyr Ala 1470 TAT TAC AAA GOA Tyr Tyr Lys Ala 1485 4416 AAG, GAT ATO CCA Lys Asp Ile Pro 1475 ATC AGG GAT TTG Ile Axg Asp Leu 1490 ACC TAC AAA Thr Tyr Lys GAA GAA Giu Glu 1480 GTA AAA TCT Vai Lys Ser 4464 COT CCA TTG TCA 'ICC TCA GAA ATG GAA GAA TTT TTA Pro Pro Leu Ser Ser Ser Giu Met Giu Giu Phe Leu 1495 1500 ACT CAG Thr Gin 1505 GAA TOT AAG AAA CAT GAA AAT GAA TTT AAT GAA GAA GTG GC Giu Ser Lys Lys His Giu Asn Glu Phe Asn Giu Giu Val Ala 1510 1515 1520 TTG ACA GAA ATT Leu Thr Glu Ile TAO Tyr 1525 AAA TAO ATO GTA AAA TAT TTT Lys Tyr Ile Val Lys Tyr Phe 1530 AAT AAA OTA GAA AGA GAA OGA GGG Asn Lys Leu Giu Arg Giu Arg Giy 1540 CTG GAA GAA GOT Leu Glu Giu Ala 1545 GAT GAG ATT OTA Asp Glu Ile Leu 1535 CAG AAA CAA OTO Gin Lys Gin Leu 1550 TGC AAG TGG ATG Cys Lys Trp Met 1565 4512 4560 4608 4656 4704 TTG OAT GTA AAA GTO TTA TTT GAT GAA AAG AAG AAA Leu His Val Lys Val Leu Phe Asp Giu Lys Lys Lys 1555 1560 TAA INFORMATION FOR SEQ ID NO:2: 4707 Wi SEQUENCE CHARACTERISTICS: LENGTH: 1569 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID KO:2: Met Giu Val Ser Arg Arg Lys Ala Pro Pro 10 Ala Pro Leu Pro Leu Leu Ala Tyr Leu Leu 25 Arg Pro Pro Arg Pro Ala Ala Leu Ala Ala Pro Gly Ile Gly Ala Arg Gly Ala Asp Giu Pro Val Trp Arg Ser Glu 4Gin Ala 40 Ile Ala Ala Ser Gin Giu Asp Gly Val Phe Val Ala Ser Gly Ser Cys WO 01118044 WO 0118044PCTUSOO/24560 Leu Arg Al a Arg Phe Asn 145 Ser Leu A-rg Asp Leu 225 Asn Ala. Val Asp Val 305 Arg so Asp Asp Arg Giu Asp 130 Ser Thr Ala Cl's Thr 210 Cl's Gil' Ala ILeu Giy 290 rrp Ser Gin Gin Pro Gil' 115 Arg Leu Ala Val Asn 195 Giu Giu Ser Thr Phe 275 Arg Ala Pro Leu Ala Arg 100 Al a Gil' Arg Gil' Ala 180 Pro Gil' Gil' Ile Gily 260 Gin Arg Thr Asp Tl'r 70 Gil' Asn Pro Gil' Ala Gil' Ala Cl's Asn Gil' 150 Val Val 165 Ala Thr Ala Ala Arg Ser Ala Gil' 230 Ty'r Phe 245 Trp Pro Gil' Gin Leu Leu Val Phe 310 Thr Thr Ser C 1 's Ser Leu Giu 135 Thr Tyr Tl'r Ser Leu 215 Ser Pro Ser Ala Leu 295 Ser Ala Leu Thr Ser Gly 120 Val1 Giu Arg Val Asp 200 Ala Leu Tyr Met Ser 280 Ser Ala Leu Giu Giu *Phe 105 *Gil' Arg Val Ala Leu 185 His Thr His Ty'r Ala 265 Leu Ser Ala Cl's His Pro 90 Ser Leu Pro Val Gil' 170 Pro Asp Gin Phe Pro 250 Arg Asp Ser Ala Leu 330 Ser 75 Val Lys Leu Leu Ser 155 Arg Glu Thr Giu Val 235 Tyr Ile Cys Leu Gly 315 Phe Leu Ser Arg Ser Leu Ala Leu Leu Leu 110 Leu Thr Gil' 125 Gil' Asn Leu 140 Cys His Pro Asn Asn Arg Pro Giu Thr 190 Ala Ile Ala 205 Leu Gil' Arg 220 Asp Ala Phe Asn Tyr Thr Ala Gin Ser 270 Gly His Gil' .285 Val Giu Ala 300 Glu Gil' Gin Arg Met Ser Leu Pro Pro Trp Ser Gin Trp 175 Ala Leu Leu Leu S er 255 Thr His Leu Giu Giu 335 Tyr Pro Tyr Thr Arg Gil' 160 Tyr Ser Lys Ly's Trp 240 Giy Glu Pro Asp Arg 320 Ile 325 Gin Ala Arg Ala Ll's Arg Val Ser 340 Asp Phe Ly's Thr Ala Giu Ser 350 WO 01/18044 His Cys Lys Giu 355 PCT/USOO/24560 Gly Asp Gin Pro Giu 360 Arg Val Gin Pro Ile Ala Ser 365 Ser Thr Leu Ile His Ser Asp Leu Thr Ser Val Tyr Gly Thr Val Val 370 375 r Met Asn Arg Thr Val Leu Phe Leu Gly Th~ 385 390 Lys Val Ile Leu Giy Giu Asn Leu 405 Tyr Giu Ile Lys Giu Giu Thr Pro 420 Pro Val Lys Asn Ile Tyr Ilie Tyr 435 440 Arg Ile Arg Val Ala Asn Cys Asn 450 455 Leu Thr Ala Thr Asp Pro His Cys 465 470 Cys Thr Phe Gin Giy Asp Cys Val 485 Leu Asp Ile Her Ser Giy Ala Lys 500 Arg Ser Ser Lys Glu Lys Thr Thr 515 520 Pro Arg His Her Lys Cys Met Val 530 535 Leu Cys Gin Asn Lys Ser Gin Pro 545 550 Pro Thr Arg Ala Thr Tyr Lys Asp 565 Ser Phe dly Ser Trp Asn Leu Ser 580 Her Her Leu Lys Giu Cys Pro Ala Thr Ser 410 Val Phe 425 Leu Thr Lys His Gly Trp, His Her 490 Lys Cys 505 Val Thr Lys Asn PAsn Arg Val Ser 570 %sp Arg 1 585 ys Val C Giy 395 Asn Tyr Ala Lys Cys 475 Glu Pro Met Val rhr '555 lal I ?he I flu T1 ~he T 3 8 ASi CY~ Lys Gi7 Ser 460 His Asn Lys Vl ksp 540 ys Tal Lsnf hr 'hr 0 SGly Gin Pro Giu Leu Val 430 Lys Giu 445 Cys Her Her Leu Leu Giu Ile Gin 510 Gly Her 525 Ser Ser Thr Cys Asn Vai b~ Phe Thr A~ 590 Gly Cys 605 Ala Cys A Let~ Val 415 Pro Val Glu Gin Asn 495 Ile Phe !Lrg ~er let Lfla Ia 1Leui 400 Ile Asp Arg Cy r Arg 480 Trp le Her Glu Ile 560 Phe Cys Trp Pro 595 600 Cys Lys Ser Ala Arg A-rg Cys Ile His Pro 610 615 P 620 Asp Tyr Giu Arg Asn 630 Gin Glu Gin Cys Val Ala Val Giu Lys 640 WO 0/18044 Thr Ser Giy Gin Ala Leu Ser Thr Leu 675 Arg Ala Ser 690 Lys Asp Val 705 Phe Ser Leu Phe Asp Gly Leu Pro His 755 Gly Gin Asn 770 Leu Ile Ile 785 Cys Val Ala Lys Val Arg Leu Asp Cys 835 Tyr Arg Val 850 Lys Giu Asn 865 Leu Phe His Thr Arg Asn Lys Thr Ala 915 Giy Gin 660 Gly Asn Ile Pro Gly 740 Cys Ile Ser Thr Thr 820 Gly Glu Asp Gly Gin 900 Ser Pro Tyr As n Met 695 Ser Arg Ser Ile Met 775 Leu Gly Thr Gin Vai 855 Asn Gly Thr Ala Lys Giu Asn 650 Ile Lys Ser 665 Vai Ile Val 680 Ile Leu Lys His Val Leu Lys Giu Met 730 Ser Val Giy 745 Phe Pro Aia 760 Giy Arg Asn Lys Giy Asn Phe Leu Ala 810 Val Lys Leu 825 Tyr Arg Giu 840 Asp Thr Glu Ile Ser Lys Gin Leu Asn 890 Thr Ile Leu 905 Asn Ser Ser 920 PCTUSOOI2456O Thr Asn 655 Lys Val Phe Thr Cys Asp Met Lys 720 Ile Gin 735 Ile Aia Ser Giy Asp Asn Giu Tyr 800 Ser Ser 815 Thr Tyr Thr Gly Ile Gin Ile Thr 880 Asn Ile 895 Gly Ile Val Lys Leu Gly Asn Leu Giu Leu Tyr Vai Giu Gin Giu Ser Vai Pro Ser Thr WO 01/18044 PCTIUSOOI24560 930 95940 Trp Tyr Phe Leu Ile Val Leu Pro Val Leu Leu Val Ile Val Ile Phe 945 950 955 960 Ala Ala Val Gly Val Thr Arg His Lys Ser Lys Giu Leu Ser Arg Lys 965 970 975 Gin Ser Gin Gin Leu Giu Leu Leu Glu Ser Glu Leu Arg Lys Glu Ile 980 985 990 Arg Asp Gly Phe Ala Giu Leu Gin Met Asp Lys Leu Asp Val Vai Asp 995 1000 1005 Ser Phe Gly Thr Val Pro Phe Leu Asp Tyr Lys His Phe Aia Leu Arg 1010 1015 1020 Thr Phe Phe Pro Giu Ser Gly Gly Phe Thr His Ile Phe Thr Giu Asp 1025 1030 1035 1040 Met His Asn Arg Asp Ala Asn Asp Lys Asn Glu Ser Leu Thr Ala Leu 1045 1050 1055 Asp Ala Leu Ile Cys Asn Lys Ser Phe Leu Vai Thr Val Ile His Thr 1060 1065 1070 Leu Giu Lys Gin Lys Asn Phe Ser Val Lys Asp Arg Cys Leu Phe Ala 1075 1080 1085 Ser Phe Leu Thr Ile Ala Leu Gin Thr Lys Leu Vai Tyr Leu Thr Ser 1090 1095 1100 Ile Leu Giu Val Leu Thr Arg Asp Leu Met Giu Gin Cys Ser Asn Met 1105 1110 1115 1120 Gin Pro Lys Leu Met Leu Arg Arg Thr Ghi Ser Vai Val Glu Lys Leu 1125 1130 1135 Leu Thr Asn Trp Met Ser Val Cys Leu Ser Gly Phe Leu Arg Giu Thr 1140 1145 1150 Val Gly Giu Pro Phe Tyr Leu Leu Val Thr Thr Leu Asn Gin Lys Ile 1155 1160 1165 Asn Lys Gly Pro Vai Asp Val Ile Thr Cys Lys Ala Leu Tyr Thr Leu 1170 1175 1180 Asn Giu Asp Trp Leu Leu Trp Gin Vai Pro Giu Phe Ser Thr Val Ala 1185 1190 1195 1200 Leu Asn Val Val Phe Giu Lys Ile Pro Giu Asn Giu Ser Ala Asp Val 1205 1210 1215 Cys Arg Asn Ile Ser Val Asn Vai Leu Asp Cys Asp Thr Ile Gly Gin 1220 1225 1230 12 WOO01/18044 PCTIUSOOI2456O Ala Lys Giu Lys Ile Phe Gin Ala Phe Leu Ser Lys Asn Gly Ser Pro 1235 1240 1245 Tyr Gly Leu Gin Leu Asn Giu Ile Gly Leu Giu Leu Gin Met Gly Thr 1250 1255 1260 Arg Gin Lys Giu Leu Leu Asp Ile Asp Ser Ser Ser Vai Ile Leu'Giu 1265 1270 1275 1280 Asp Gly Ile Thr Lys Leu Asn Thr Ile Gly His Tyr Giu Ile Ser Asn 1285 1290 1295 Gly Ser Thr Ile Lys Val Phe Lys Lys Ile Ala Asn Phe Thr Ser Asp 1300 1305 1310 Val Glu Tyr Ser Asp Asp His Cys His Leu Ile Leu Pro Asp Ser Glu 1315 1320 1325 Ala Phe Gin Asp Val Gin Gly Lys Arg His Arg Gly Lys His Lys Phe 1330 1335 1340 Lys Val Lys Giu Met Tyr Leu Thr Lys Leu Leu Ser Thr Lys Val Ala 1345 1350 1355 1360 Ile His Ser Val Leu Glu Lys Leu Phe Arg Ser Ile Trp Ser Leu Pro 1365 1370 1375 Asn Ser Arg Ala Pro Phe A-la Ile Lys Tyr Phe Phe Asp Phe Leu Asp 1380 1385 1390 Ala Gin Ala Glu Asn Lys Lys Ile Thr Asp Pro Asp Val Val His Ile 1395 1400 1405 Trp Lys Thr Asn Ser Leu Pro Leu Arg Phe Trp Val Asn Ile Leu Lys 1410 1415 1420 Asn Pro Gin Phe Vai Phe Asp Ile Lys Lys Thr Pro His Ile Asp Gly 1425 1430 1435 1440 Cys Leu Ser Val Ile Ala Gin Ala Phe Met Asp Ala Phe Ser Leu Thr 1445 1450 1455 Glu Gin Gin Leu Gly Lys Giu Ala Pro Thr Asn Lys Leu Leu Tyr Ala 1460 1465 1470 Lys Asp 1ie Pro Thr Tyr Lys Giu Giu Val Lys Ser Tyr Tyr Lys Ala 1475 1480 1485 Ile Arg Asp Leu Pro Pro Leu Ser Ser Ser Giu Met Glu Giu Phe Leu 1490 1495 1500 Thr Gin Giu Ser Lys Lys.His Glu Asn Giu Phe Asn Giu Giu Val Ala 1505 1510 1515 1520 WO 01/18044 PCTIUSOO/24560 Leu Thr Glu Ile Tyr Lys Tyr Ile Val Lys Tyr Phe Asp Glu Ile Leu 1525 1530 1535 Asn Lys Leu Giu Arg Giu Arg Gly Leu Giu Giu Ala Gin Lys Gin Leu 1540 1545 1550 Leu His Val Lys Val Leu Phe Asp Glu Lys Lys Lys Cys Lys Trp Met 1555 1560 1565