WO2001018044A2 - Use of semaphorin for modulation of cellular efflux - Google Patents

Abstract

The present invention is directed to compositions and methods comprising the use of semaphorins, semaphorin receptors, and polynucleotides encoding semaphorins and semaphorin receptors, for the modulation of cellular efflux mumps. Included in the present invention are compositions and methods for control of multiple drug resistance phenotypes via control of cellular efflux.

Description

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 semaphonn or semaphorin receptor polypeptides, as well as polynucleotides encoding these polypeptides, to modulate cellular efflux or the MDR phenotype of cells. π. 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 Multidrug 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 chloride ions. For example, MDR protein pumps include the proteins MDR-1 and MDR-2, which are each considered to be a P-glycoprotein (P-gp), 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 P- gp 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, e.g., 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.

HI. 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 immuno-deficiency disorders such as HIV, and other cellular-efflux-related disease states. 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 semaphonns 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:l 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 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. 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 administer in conjunction with the disclosed compositions include virtually any chemotherapeutic or sensitizing agent such as cyclosporin, FK506, taxotere, doxorubicin, cis-platin, tamoxifen, i- phosphamide, or mefhotrexate, 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, α-interferon, β-interferon, or y- interferon. 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: (1) 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; (3) observing the rate of death of a control cell in the absence of the test compound; and (4) 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 i- phosphamide. 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: (1) contacting a first cell with a test compound and a semaphorin or semaphorin receptor, in the presence of a dye; (2) measuring the net rate of influx of dye into the first cell; (3) 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 (4) comparing the net rate influx of dye into the first cell to the net rate of 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 BODIPY- transferrin.

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: (1) to increase vulnerability or sensitivity of a cell to cytotoxic agents and thereby promote drug-induced cell death; (2) in identification or design of semaphorin or semaphorin receptor antagonists or agonists that might increase the sensitivity of a cell to a cytotoxic agent; (3) to promote cellular resistance to cytotoxic agents; or (4) in identification of semaphorin or semaphorin receptor agonists or antagonists that can be administered to cells to promote their resistance to various cytotoxic substances. 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; CD 100; 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. 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:l; and those segments of SEQ ID NO: l 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, e.g., by centrifugation, and assaying the medium (supernatant) 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 fragments advantageously include regions that are conserved in the 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 30, 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 (J. 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: (1) a scoring matrix, blosum62, as described by Henikoff and Henikoff (Proc. Natl. Acad. Sci. USA 89:10915, 1992); (2) a gap weight of 12; (3) a gap length weight of 4; and (4) 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 of mRNA 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. 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, e.g., 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 He, 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 native- pattem glycosylation. Polypeptides expressed in yeast or mammalian expression systems (e.g., COS-1 or COS-7 cells) can be similar to or significantly different from a native polypeptide in molecular 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 non- glycosylated 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 non- covalently-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. 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, e.g., by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn 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 IgGl 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 IgGl 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 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, e.g., Lelong, et al., 1991). One example of use of these dyes involves loading the dye into a cell at low temperatures, such 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 BODIPY- transferrin (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. 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: (1) contacting a first cell with a test compound including a semaphorin or semaphorin receptor in the presence of a dye; (2) measuring the net rate of influx of dye into this first cell; and (3) 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, CD 100, 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 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 of MDR 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 e«v - that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene, termed ψ, 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 ψ components is constructed (Mann et al, 1983). When a recombinant plasmid containing an inserted DNA, together with the retroviral LTR and ψ sequences, is introduced into this cell line (by calcium phosphate precipitation for example), the ψ 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 etal, 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 of adenovirus 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 El A and E1B 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-Perricaudet 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. Ad5 -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; (v) 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 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 α/., 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 al, 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 al, 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 (Y^ang ^et l. , 1990), polycations (Boussif et al , 1995) and receptor-mediated transfection (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 al (1984) successfully injected polyomavirus DNA in the form of CaP0₄ 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 CaP0₄ 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 al, 1990; Zelenin et al, 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 of MDR 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 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 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 lOOng/ml to about lOOμg/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 1 μg/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 re- seal 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. 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.

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 al, Molecular Pharmacology 40:490 (1991). Schindler, et al, Biochemistry 35:2811 Weisburg et al, J. Biol Chem, 274, 10877-88 (1999) Yamada et.al, PNAS 94:14713-14718 (1997) Zaman, G.J.R. et.al, PNAS USA 91:8822 (1994)

Claims

CLAIMSWhat is claimed is:

1. Use of a semaphorin, a semaphorin receptor, or an agonist or antagonist of a semaphorin or semaphorin receptor, in the manufacture of a medicament for multiple drug resistance, cystic fibrosis or immunodeficiency disease.