USE OF THE INTERFERON RECEPTOR 2c POLYPEPTIDE CHAIN TO ENHANCE THE ANTI-GROWTH EFFECTS OF TYPE I INTERFERONS

Abstract: The present invention is directed towards a method of potentiating the anti-growth effects of type I interferon (IFN) on cells in a target cell population comprising increasing the number of functional IFNAR2c receptors on the surface of modified cells within the target cell population and then exposing the modified cells to a therapeutically effective amount of a type I IFN or by exposure to endogenously produced IFN.

Description: USE OF THE INTERFERON RECEPTOR 2c POLYPEPTIDE CHAIN TO ENHANCE THE ANTI-GROWTH EFFECTS OF TYPE I INTERFERONS This application claims benefit of U.S. Provisional Application No. 5 60/220,844, filed July 26, 2000, which is incorporated by reference in its entirety. BACKGROUND OF THE INVENTION Since their initial discovery in 1957, interferons, originally described as factors that interfered with viral infection, have been widely studied. Interferons (TFNs) are 0 now recognized as an integral part of the body's natural defense system, and they are used successfully as therapeutic agents for the treatment of a number of human diseases. IFNs are segregated into two classes defined as either type I or type II. Type I IFNs include a family of related proteins, IFN , IFN , IFN , IFN and lPN , whereas type π IFN consists of one protein, IFN , which has limited homology to 5 type I IFNs. Two receptor proteins, IFNAR1 and IFNAR2 are known to be involved in type I IFN binding. DESCRIPTION OF THE INVENTION o The present invention relates to methods of potentiating the effects of effector ligands by increasing the number of functional receptors for receptor ligands on the cell surface. As used herein, increasing the number of functional receptors means any amount of expression of functional receptor in excess of the amount normally expressed by the cell. Thus, the increase may be accomplished by increasing the total 5 number of receptor proteins on the surface of the cell or by replacing non-functional receptor protein with functional receptor protein, such as, for example, by mutagenesis. "Potentiate", as used herein, means any enhancement of the effects of the effector ligand. In one aspect, the present invention relates to a method of potentiating the o effects of an effector ligand on a target cell population comprising tumor cells by increasing the number of functional receptors for the effector ligand on the cell surface of modified cells within the target cell population and then treating the modified cells with a therapeutically effective amount of the effector ligand. As used herein, a target cell population may comprise one or more cells and modified cells within the target cell population may comprise one or more cells of the target cell population. In another aspect, the present invention relates to a method of potentiating the 5 effects of IFN on a target cell population by increasing the number of functional receptors for IFN on the cell surface of modified cells within the target cell population and then treating the modified cells with a therapeutically effective amount of IFN. Effects of IFN include, for example, antiviral effects, anti-growth effects, and immunoregulatory effects. For example, it has been found that increasing the o expression of the IFNAR2c polypeptide on the surface of cells within a target cell population potentiates the effects of type I IFN on cells of the target population. In particular, increasing the number of functional receptors for type I IFN on the cell surface of modified cells within a target cell population can potentiate the anti-growth effects of type I IFN on the target cell population. 5 As used herein, anti-growth effects include anti-proliferative and apoptotic effects, as well as any other effects which result in cell death, a cessation of cell growth, or the slowing of cell growth. Anti-proliferative effects, as used herein, includes, for example, cell cycle arrest or an increase in cell cycling time, as well as the induction of inducers of apoptosis, the induction of factors negatively regulating o protein synthesis, DNA synthesis, or RNA synthesis, or the activation of inhibitors of metabolic pathways. Without wishing to be limited by any theory of the invention, the inventors believe that the type I IFN acts directly on cells with mcreased JJFNAR2c expression to elicit these anti-growth effects. However, it is also contemplated as part of this 5 invention that, in lieu of the direct effect or in addition to the direct effect, type I IFN may exhibit anti-growth effects via a bystander effect, wherein the type I IFN acts on the cells with increased IFNAR2c expression to elicit the secretion of a factor which has an anti-growth effect on adjacent cells. Accordingly, the present invention relates to methods of potentiating the anti- o growth effects of a type I IFN on a target cell population by increasing the number of functional IFNAR2c receptor chains expressed on the surface of modified cells within the target cell population and then treating the modified cells with a therapeutically effective amount of at least one effector ligand which binds to the type I IFN receptor. Increasing the number of functional IFNARl receptor chains on the surface of the cell is also contemplated as part of this invention. A preferred effector ligand which binds to the type I JJFN receptor is a type I IFN. "Modified cells", as used herein, means cells which have been modified to express increased levels of a functional receptor for an effector ligand on the cell surface. Modified cells may include cells that have been modified in vivo or ex vivo to express increased levels of the functional receptor for the receptor ligand. Modified cells which are modified ex vivo can be subsequently transferred to the target cell 0 population after ex vivo modification. Modified cells may also include naturally occurring cells which express relatively high levels of the effector ligand receptor and which are added to the target cell population. As used, herein, an effector ligand refers to any molecule which binds to the effector ligand receptor and which achieves at least partial activation of the effector 5 ligand receptor. Effector ligands include, but are not limited to, naturally occurring effector ligands, modified effector ligands, chimeric effector ligands, effector ligand mimetics, or antibodies to the effector ligand receptor. Preferred effector ligands include, for example, growth factors, cytokines, chemotactic factors, and hematopoietic factors. Particularly, preferred effector ligands o include, but are not limited to, the following: IFNs; tumor necrosis factors (TNF), for example, TNF and TNF ; interleukins (EL), for example, IL-1, EL-2, JJL-3, IL-4, IL-5, IL-6, IL-7, ΓL-8, IL-9, IL-10, ΓL-I I, IL-12, and IL-13; colony stimulating factors (CSF), for example, granulocyte-macrophage-CSF (GM-CSF), monocyte-CSF (M- CSF), granulocyte-CSF (G-CSF); erythropoietin (EPO); stem cell factor (SCF); 5 leukemia inhibitory factor (LIF); epidermal growth factor (EGF), Oncostatin M (OSM), chemokine receptor 1 or 5 (CCR1 or CCR5), etc. A particularly preferred effector ligand is an effector ligand which binds to a type I IFN receptor, including type I IFNs. As used herein type I IFNs include IFN , IFN , IFN , IFN and IFN , or any newly defined type I IFN, all of which may be o used in accordance with the invention, hi accordance with the invention, the type I IFN employed maybe, for example, any subtype with anti-growth activity, antiviral activity, or immunoregulatory activity. The subtype may be a naturally occurring or a recombinant subtype, including hybrids of two or more subtypes, or analogs thereof. Anti-growth activity may be determined by any techniques that are well known in the art, including those described in the examples below. Methods in Enzymology, Vol. 119, ed. Sidney Pestka. A type I IFN used in accordance with this invention 5 preferably has an anti-growth activity such that thymidine incorporation in HT1080 cells is inhibited by 5%, preferably 10%, more preferably 15% after incubation for 24 hours in an optimal concentration of the IFN. Mixtures of different subtypes may also be used. Preferably, the type I IFNs that may be used in accordance with this invention include IFN and IFN , including but not limited to the following: IFN o subtypes, including but not limited to IFN lb and IFN 1 a; IFN subtypes including but not limited to IFN 2, IFN 5, TEN 13, TEN 6, IFN 14, IFN 16, IFN 21, IFN 10, TEN 46, IFN 46, IFN 7, and consensus alpha IFNs. Examples of IFNs that may be used in conjunction with the invention include but are not limited to the following. For IFN lb, "Betaseron", a recombinantly produced human TEN , 5 wherein the cysteine residue at the 17 position has been replaced by serine, as disclosed and claimed in U.S. Patent No. 4,588,585, may be employed. Additionally, a recombinantly produced IFN la, which is produced in Chinese hamster ovary (CHO) cells, may also be employed. For IFN , human alpha-TEN products Intron® (Schering-Plough), Roferon® (Hoffman-LaRoche) and Infergen® (Amgen) maybe o employed. Other IFNs which may be employed include consensus type I alpha IFNs as described, for example in U.S. Patent Nos. 4,695,623, 4,897,471, and 5,541,293. Interferons employed as part of this invention may also be modified by conjugation to other molecules, such as described for example, in U.S. Patent No. 5, 981,709. It is contemplated as part of this invention that the number of functional 5 effector ligand receptors on the surface of a modified cell may be increased in a variety of different ways. For example, up-regulation of gene expression of the IFNAR2c gene may be employed to increase the number of IFNAR2c receptor proteins on the cell. Up-regulation of gene expression maybe accomplished, for example, by introducing an exogenous polynucleotide encoding the IFNAR2c o polypeptide into the modified cells or by positively affecting gene transcription of the endogenous IFNAR2c gene or an exogenous IFNAR2c gene in the modified cells. For example, up-regulation of gene expression may be accomplished by stimulating the promoter or other regulatory sequences, either directly or indirectly, or by activating genes which stimulate TENAR2c polypeptide production. For example, small molecules may be employed to stimulate the promoter of the TENAR2c gene. Small molecules that up-regulate transcription may be identified using techniques that 5 are well known in the art. Additionally, various methods that are well known in the art may be employed to increase the stability of messenger RNA coding for the IFNAR2c protein or to increase stability of the IFNAR2c polypeptide. A preferred method for increasing the number of functional TENAR2c polypeptides on the surface of modified cells is by the introducing at least one 0 exogenous polynucleotide encoding an IFNAR2c polypeptide into the modified cells. Any exogenous TENAR2c gene may be used in accordance with the invention, including, for example, the human TENAR2C gene or any other mammalian TENAR2c gene such as mouse, rat, etc. The DNA sequence of a gene encoding the full-length human TENAR2c polypeptide, and the corresponding amino acid sequence, are 5 described in Lutfalla et al., 1995, EMBO J. 14(20):5100-5108; Domanski et al., 1995, J. Biol. Chem. 270:21606-21611; and SEQ TD NOS. 3 and 4, filed herewith. The DNA sequence of a gene encoding the full-length murine TENAR2c polypeptide, and the corresponding amino acid sequence are shown in Owczarek et al., 1997, J. Biol. Chem. 272:23865-23870 and SEQ ID NOS. 1 and 2, filed herewith. A partial DNA o sequence of the gene encoding the human TENAR2c polypeptide are also disclosed in Novick, U.S. Patent No. 5,821,078, EP 0 588 177 A2, and EP 0676 413 A2. As used herein, an exogenous TENAR2c gene refers to any IFNAR2c gene which is added to the cells, including the addition to the cell of another copy of any endogenous TENAR2c gene. 5 The invention relates not only to increasing the number of naturally occurring JJFNAR2c polypeptides on the surface of a target cell, but also to increasing the number of mutant IFNAR2c polypeptides on the surface of a cell. For example, changes in the amino acid sequence of TENAR2c are contemplated in the present invention. The TENAR2c polypeptide can be altered by changing the DNA encoding o the protein. Conservative amino acid sequence alterations are preferred, using amino acids that have the same or similar properties. Illustrative amino acid substitutions include the following changes: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, glutamine, or glutamate; methionine to leucine or isoleucine; phenylalanine 5 to tyrosine, leucine or metMonine; serine to threonine; threonine to serine; tryptophan to tyrosme; tyrosine to tryptophan or phenylalanine; valine to isoleucine or leucine. Polynucleotide sequences of the invention include DNA, cDNA and RNA sequences which encode IFNAR2c polypeptides. Such polynucleotides include naturally occurring, synthetic, and intentionally manipulated polynucleotides. For o example, portions of the mRNA sequence may be altered due to alternate RNA splicing patterns or the use of alternate promoters for RNA transcription. As another example, IFNAR2c polynucleotides maybe subjected to site-directed mutagenesis. The invention includes any polynucleotide encoding an IFNAR2c polypeptide having biological activity. Biological activity of an IFNAR2c polypeptide, as used herein, 5 refers to a polypeptide which, when expressed on the surface of a cell, has one of the following activities: (1) binds a type I TEN; (2) is able to potentiate at least some TEN- mediated activity when dimerized with IFNAR1 ; (3) antibody binding activity, wherein antibodies directed against IFNAR2c inhibit or block at least one IFN- mediated activity. o Suitable target cell populations for the practice of the invention include any cell population which would benefit from a potentiation of TEN-mediated activity. For example, prohferative cell conditions may be treated in accordance with this invention. Prohferative cell condition, as used herein, includes any condition wherein it is desirable to limit cell proliferation, including cancer or neoplastic conditions. 5 Cancer cells include but are not limited to cells involved in hairy cell leukemia, multiple myeloma, Kaposi's sarcoma, cervical neoplasia, basal cell carcinoma, squamous cell carcinoma, melanoma, renal cell carcinoma, carcinoid tumors, cutaneous T cell lymphoma, non-Hodgkins's lymphoma, head and neck tumors, and breast, lung and prostate tumors, pancreatic tumors, and adenocarcinomas. Cancer o cell populations which are part of solid tumors are particularly contemplated as part of this invention. Additionally, inoperable cancers such as brain cancer, pancreatic cancer, and later stage metastatic disease are particularly contemplated as part of the invention. Cells involved in prohferative cell conditions include cell types involved in myeloprohferative disorders. Examples of myeloproliferative disorders include, for 5 example, chronic myelogenous leukemia, polycythaemia vera, agnogenic myeloid metaplasia and idiopathic thrombocythaemia. Prohferative cell conditions also include conditions, such as, for example, coronary restenosis. The use of type I TENs for the treatment of coronary restenosis is described for example in U.S. Patent No. 5,681,558. 0 It is also contemplated as part of this invention that the methods of this invention may be used in conjunction with the administration of other growth regulator polypeptides to the target cell population. For example, according to the methods of the invention, TEN may be administered to the target cell population containing cells modified to express increased levels of an IFNAR2c polypeptide in 5 conjunction with the administration to the target cell population of other growth regulator polypeptides such as growth factors or cytokines, including for example interleukins. Additionally, in accordance with the invention, cells of the target cell population maybe modified to express increased amounts of more than one type of effector ligand receptor, such as for example, a type I TEN receptor and an interleukin o receptor, either on the same modified cells or different modified cells within a target cell population, with subsequent treatment with the corresponding effector ligands. The present invention also provides gene therapy for the treatment of prohferative cell conditions. Such therapy would achieve its therapeutic effect, for example, by introduction of the TJFNAR2c gene into modified cells of the target cell 5 population, followed by treatment of the modified cells with a type I TEN or any other effective ligand to the TEN receptor. The ligand to the TEN receptor may be added exogenously or produced endogenously. The TENAR2c gene may be delivered to the organism in any effective manner, e.g. using a vector or other delivery vehicle, or as naked DNA. DNA delivery vehicles can include viral vectors such as adenoviruses, o adeno-associated viruses, and retroviral vectors. See, for example: Bilbao et al., 1998, Tumor Targeting 3:59-79; Yia-Herttuala and Martin, 2000, Lancet 355:213- 222; Chu et al., 1994, Gene Therapy 1:292-299; Couture et al., 1994, Human Gene Therapy 5:667-677; Eiverhand et al, 1995, Gene Therapy 2:336-343. Non-viral vectors which are also suitable include DNA-lipid complexes, for example liposome mediated or ligand/poly-L-lysine conjugates, such as asialoglyco-protein-rnediated delivery systems. See, for example: Feigner et al., 1994, J. Biol. Chem. 269:2550- 5 2561; Derossi et al., 1995, Restor. Neurol. Neuros. 8:7-10; and Abcallah et al., 1995, Biol. Cell 85:1-7. Retro viruses from which the retro viral vectors may be derived include but are not limited to Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). It is also contemplated as part of this invention that the o ligand, or a gene encoding the ligand, may be included in the gene therapy delivery vehicle along with the gene encoding the IFNAR2c polypeptide. The vector is administered to the host either locally or systemically. Typically, the vector is administered systemically by intravenous injection. Suitable viral titers will depend on a number of factors, such as the particular vector chosen, the host, the 5 mode of administration, the strength of the promoter used, and the severity of the disease being treated. For mice, an adenovirus vector is preferably administered as an injection at a dose range of from about 5.0 x 10 to about 10 x 10 plaque forming units (PFU) per gram body weight. Preferred dosages range from at least about 6-9 x 10 PFU per gram of body weight, and more preferred is from at least about 6.7-8.6 x 0 10 PFU per gram of body weight. Animals which contain modified cells that express increased levels of the TENAR2c gene are useful models for studying the effect of type I TEN on target cell populations that contain the modified cells. This invention is specifically directed to gene therapy in humans. Also contemplated as part of this invention is the use of gene 5 therapy in animals, including household pets and farm animals. Administration of a type I TEN, or other ligand, in pure form or in an appropriate pharmaceutical composition can be carried out via any of the accepted modes of administration. The type I TEN may be administered locally or systemically. Thus administration can, for example, be orally, nasally, parenterally, topically, o transdermaliy, or rectally. The type I TEN may be administered as solid or semi-solid dosage forms, lyophilized powder, or liquid dosage forms, including for example, tablets, suppositories, pills, soft elastic and hard gelatin capsules, powders, solutions, suspensions, or aerosols, or the like, preferably in unit dosage forms suitable for simple adiriinistration of precise dosages. The pharmaceutical conditions will typically include a conventional pharmaceutical carrier or excipient and the type I TEN. The compositions may also include other medicinal agents, pharmaceutical 5 agents, carriers, adjuvants, etc. For a brief review of present methods for drug delivery, see Langer, 1990, Science 249:1527-1533, which is incorporated herein by reference. The preferred route of administration of the type I TEN is parenterally, using a convenient daily dosage regimen. For such parenteral administration for example, a o pharmaceutically acceptable composition containing a human -TEN may be formulated by methods disclosed in U.S. Patent Nos. 4,462,940, 4,588,585 and 4,992,271. By "therapeutically effective amount", as used herein, is meant the quantity of a type I TEN sufficient to induce anti-proliferative effects in the target cell population. 5 Amounts effective for this use will, of course, depend on the condition being treated and the weight and general state of the subject. Various considerations are described, e.g., in Gilman et al. (eds.) (1990) Goodman and Gilman 's: The Pharmacological Bases of Therapeutics, 8th ed., Pergamon Press; and Remington 's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, PA, each of which is herein o incorporated by reference. A therapeutically effective dose of a type I -TEN in a human is typically, for example, about 0.05 mg Betaseron to about 0.25 mg Betaseron, administered subcutaneously, every other day. Therapeutically effective amounts of other interferon products, including other -TEN products, may be determined routinely by 5 one of skill in the art. It is also contemplated as part of this invention that a gene encoding an IFNAR2C polypeptide will be delivered to the target cell population in conjunction with gene encoding a type I TEN. The IFNAR2c gene and the type I IFN gene may be delivered to the same cells or different cells of the target population. The TENAR2c o gene may delivered in the same composition and/or the same vector as the type I TEN gene. For example, the type I TEN gene and the TENAR2c gene may be delivered as part of the same viral vector, for example, an adenovirus vector. Additionally, it is also contemplated as part of this invention that the IFN gene may be delivered to cells that are adjacent to cells of the target population. Additionally, type I TEN may be delivered to the target cells by the implantation of cells expressing type I TEN in or near the target cell population. BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a series of histograms showing that cells transfected with the ΓFNAR2C gene express elevated levels of high affinity type I TEN receptors. Data represents the average of three separate points and standard errors are less than 10 percent of the average. Non-specific binding was measured in the presence of a 100- fold excess of unlabeled TEN. Panel A shows results obtained with transfected HT1080 cells. Panel B shows results obtained with transfected MDA231 cells. Panel C shows results obtained with U5A cells, which do not express TENAR2c, including cells transfected with truncation mutants of the IFNAR2c gene. Figure 2 is a histogram showing that U5 A cells that were rescued by transfection with a functional TENAR2c gene are extremely sensitive to the anti-proliferative activities of TEN lb in comparison to untransfected HT1080 cells. Data represents the average of three separate points and standard deviations were less than 15 percent of the average. Figure 3 is a histogram showing that HT1080 cells and HT1080 cells transfected with the TENAR2c gene (HTbetaL.2) were sensitive to both incubation times with TEN lb and concentrations of TEN lb, although HTbetaL.2 cells showed enhanced sensitivity. Figure 4 is a histogram showing a comparison of the anti-growth effects of 5000 international Units ("TU")/ml of human TEN lb (Betaseron) or human TEN 2 on HT1080 cells and HT1080 cells transfected with the TENAR2c gene, using a thymidine incorporation assay. Data is expressed as fold inhibition of cell growth. Figure 5 is a histogram showing a comparison of the anti-growth effects of TEN lb and TEN 2 on HT1080 cells transfected with the TENAR2c gene using an Alamar Blue assay. Figure 6 shows photographic images of IFNAR2c transfected HT1080 5 (HTbetaL.2) cells after treatment of the cells with TEN lb. Panels A through D are as follows: Panel A, untreated cells (10X magnification); Panel B, treated cells (10X magnification); Panel C, treated cells (20X magnification); and Panel D, treated cells (40X magnification). Cells were treated with 500 IU/ml of TEN lb. Figure 7 shows photographic images of IFNAR2c transfected MDA231 cells o after treatment of the cells with TEN lb. Panels A and B show untreated and treated parental MDA231 cells, respectively, at 10X magnification. Panels C and D show treated and untreated TENAR2c transfected MDA231 cells, respectively, at 10X magnification. Cells were treated with 500 IU/ml of TEN lb. Figure 8 is a series of microscopic images that show the results of an apoptosis 5 assay (TTJNEL assay) of the effects of TEN lb treatment of HT1080 cells that were transfected with a gene encoding the TENAR2c receptor protein (HTbetaL.2 cells). Panels A-D are as follows: Panels A and B, untreated HTbetaL.2 cells, brightfield view and fluorescent microscope view, respectively; Panels C and D, TEN lb treated HTbetaL.2 cells, brightfield view and fluorescent microscope view, respectively; o Panels E and F, parental HT1080 cells, brightfield view and fluorescent microscope view, respectively. Brightfield images Panels A, C, and E represent greater than 80% confluent cell layer. FITC labeled nucleotides were used to label DNA fragments characteristic of apoptotic cells (Panels B, D, and F). Cells were treated with 500 IU/ml of TEN lb. Magnification for Panels A-F was 10X. 5 Figure 9 is a series of microscopic images that show the results of an apoptosis assay (TUNEL assay) of the effects of TEN lb treatment of MDA231 cells that were transfected with a gene encoding the IFNAR2c receptor protein (MDAbetaL.2 cells). Panels A-F are as follows: Panels A and B, untreated MDAbetaL.2 cells, brightfield view and fluorescent microscope view, respectively; Panels C and D, TEN lb treated o MDAbetaL.2 cells, brightfield view and fluorescent microscope view, respectively; Panels E and F, parental MDA231 cells, brightfield view and fluorescent microscope view, respectively. Brightfield images Panels A, C, and E represent greater than 80% confluent cell layer. FITC labeled nucleotides were used to label DNA fragments in apoptotic cells (Panels B, D, and F). Cells were treated with 500 IU/ml of TEN lb. Magnification for Panels A-F was 10X. Figure 10 is a histogram that shows the in vivo anti-growth activity of TEN lb (Betaseron) on LOX human melanoma cells and on LOX-TENAR2c cells (LOX cells transfected with a functional TENAR2c gene) using a mouse lung seeding model. The LOX-IFNAR2c cells showed enhanced sensitivity to 200 μg Betaseron given intraperitioneally every other day. Without further elaboration, it is believed that one skilled in the art can, using the preceding description, utilize the present invention to its fullest extent. The following preferred specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in anyway whatsoever. In the foregoing and in the following examples, all temperatures are set forth uncorrected in degrees Celsius; and unless otherwise indicated, all parts and percentages are by weight. The entire disclosure of all patents, patent applications, and publications, cited above or below are hereby incorporated by reference. E X A M P L E S Example 1. Cells transfected with an TENAR2c gene express elevated levels of high affinity type I TEN receptors. Three cell lines transfected with TENAR2c genes were evaluated for expression of high affinity type I TEN receptors by ligand binding assays using a phosphorylated form of TEN 2. High affinity ligand binding has previously been demonstrated to occur only when both TENARl and TENAR2c are expressed on the cell surface (Russell-Harde et al., 1995, J. Biol. Chem. 270:26033-26036). Therefore, ligand binding was used to confirm the expression of IFNAR2c receptor protein on the surface of cell lines transfected with IFNAR2c genes. The cell lines employed were as follows: (i) HT1080 cells. A human lung fibrosarcoma cell line (ATCC No. CCL-121). 5 (ii) U5A cells. U5A cells are derived from HT1080 and are HT1080 cells that were selected for lack of TENAR2c protein expression. U5A cells lack of the ability to respond to type I TEN (Pellegrini, et al., 1989, Mol. Cell. Biol. 9:4605-4612; LutfaUa et al., \996, EMBO J. 14:5100-5108. (iii) MDA231 cells. A human breast epithelial adenocarcinoma cell line (ATCC No. 0 HTB-26). HT1080 and MDA231 cell lines were purchased from the American Type Tissue Culture (ATCC), and all cell lines were grown at 37°C in 5% CO 2 . Cells were transfected using Superfect (Qiagen Inc.), with expression plasmids containing full-length or mutant forms of human TENAR2c. Plasmids containing 5 genes encoding IFNAR2c truncation mutations or a tyrosine to phenylalanine mutation were constructed as previously described (Domanski et al., 1997, J. Biol. Chem. 272:26388-26393; Nadeau et al., 1999, J. Biol. Chem. 274:4045-4052). Stable transfected cell lines were selected in G418 (1.0 mg/ml). After selection, individual clones were picked and expanded, and integration of the IFNAR2c gene was o confirmed by PCR analysis using intron spanning primers specific for TENAR2c cDNA. Positive clones were further expanded and tested for their ability to bind type I TEN. Ligand binding assays were performed as essentially described in Croze et al., 1996, J. Biol. Chem. 271:33165-33168. TEN with a specific activity 3.0 x 10 8 IU/mg 5 was obtained from PeproTech Inc. (Rocky Hill, NJ) and the TEN 2 ligand was phosphorylated to a specific activity of 60-62 μCi/μg as previously described (Croze et al, 1996, J. Biol. Chem. 271 :33165-33168). Ligand binding was analyzed by the addition of phosphorylated TEN 2 (at a concentration of 176 pM) to 200,000 cells for ninety minutes. Non-specific binding was determined by adding 100-fold excess of o unlabeled TEN 2. Binding data were analyzed by Scatchard analysis. Parental HT1080 cells bound TEN 2 with relatively high affinity (Kd~290 pM) and 9,000 receptor cites per cell were measured by Scatchard analysis. Three stable clones of HT1080 cells transfected with the wild-type TENAR2c gene (HTbetaL.l, HTbetaL.2, and HTbetaL.4) were analyzed for TEN 2 binding. Two of the clones, 5 HTbetaL.1 and HTbetaL.2, bound ~ 8-fold more ligand (34-37 pM bound) than parental HT1080 cells, whereas the third clone (HTbetaL.4) bound approximately two-fold more ligand (11-12 pM bound) than parental HT1080 cells (Figure 1, Panel A). Cells of the human breast adenocarcinoma cell line MDA231 were also 0 transfected with a wild-type TFNAR2c gene. Two stable clones, MDAbetaL.l and MDAbetaL.2, were analyzed for TEN 2 binding. The parental MDA231 cell line bound relatively low levels of TEN 2 (2-3 pM). Each of the transfected clones bound approximately ten-fold more TEN 2 than the parental MDA231 cell line (Figure 1, Panel B). 5 U5A cells, which do not express the IFNAR2c receptor protein, were transfected with a wild-type TENAR2c gene and four mutated genes, including three truncation mutants (R2.462, R2.417, R2.346) and a full tyrosine to phenylalanine substitution mutation (R2.Y-F) (a substitution of tyrosines at positions 269, 306, 316, 318, 337, 411 and 512 of SEQ TD NO. 3 with phenylalanine). U5A cells transfected o with a wild-type IFNAR2c gene bound 13-fold more TEN than the parental HT1080 cells (Figure 1, Panel C). Stable clones expressing the either the R2.462 or the R2.346 truncation mutation, or the (R2.Y-F) deletion mutation, bound TEN 2 at levels 5 to 10-fold greater than HT1080 cells (Figure 1, Panel C). Stable clones expressing the R2.417 truncation mutation bound TEN 2 at approximately the same level as 5 HT 1080 cells (-9000 binding sites). Example 2. Enhancing the sensitivity of U5A and HT1080 cells to the anti-proliferative effects of TEN lb by transfecting the cells with an IFNAR2c gene. U5 A cells that were transfected with wild-type and mutant TENAR2c genes o were tested for sensitivity to the anti-proliferative effects of TEN lb in comparison to U5A cells. HT1080 cells that were transfected with a wild-type TENAR2c gene were also tested for sensitivity to the anti-proliferative effects of TEN and TEN in comparison to HT1080 cells. Cells were tested for sensitivity to the anti-proliferative effects of TEN lb using a thymidine incorporation assay. Cells were seeded in 24-well culture plates at a density of 2 x 10 4 cells/well, 1 ml well, and incubated with human TEN lb at a concentration of 1000 IU/ml for 24 hours. Human TEN lb (specific activity 2.5 x 10 7 IU/mg) was produced as described in (Russell-Harde, 1995, J. Interferon Cytokine Res. 15:31-37). At time zero, complete media containing tritiated thymidine ([methyl- Hjthymidme, specific activity = 40-60 Ci/mmol, Amersham Life Sciences) was added. Tritiated thymidine incorporation was measured after ten hours by the following method. Cells were washed with phosphate-buffered saline, followed by 10% trichloroacetic acid (TCA), and then 100% ethanol. Prior to the determination of the incorporation of radioactivity, cells were solubilized in 1 M potassium hydroxide and the solubilized cells were mixed with Ecolume scintillation fluid for measurement of tritiated thymidine incorporation. U5A cells that were transfected with the wild-type TENAR2c gene were extremely sensitive to the anti-proliferative activities of TEN lb whereas U5A cells were not (Figure 2). U5A cells that were transfected with a mutant TENAR2c gene that encodes a protein truncated at residue 462 were also sensitive to the anti-proliferative effects of TEN lb. Two other truncation mutants, R2.417 and R2.246, and the R2.Y-F mutant (described in Example 1 above) were unresponsive to type I TEN. Therefore, truncation of the TENAR2c receptor past residue 417 or removal of all the tyrosines present in the intracellular region of IFNAR2c (the R2.Y- F mutant) renders cells containing the two type I TEN receptor proteins insensitive to the anti-proliferative effects of type I TENs. However, removal of the distal fifty-three residues (R2.462) of the TENAR2c protein apparently has no effect on the receptor mediated anti-proliferative effects of type I TENs. HT1080 cells expressing normal levels of the TENAR2c receptor protein are only weakly sensitive to the anti- prohferative activities of TEN lb. TABLE I 1. Anti-proliferation was measured by thymidine incorporation assay. 2. Cell death was measured by visual inspection as described in Example 4 below. 3. Apoptosis was measured by TUNEL assay, as described in Example 5 below. Example 3. IFNAR2c transfected HT1080 cells expressing enhanced levels of TENAR2c were sensitive to the effects of both type I TEN and type I TEN . The effects of varying incubation times and concentrations of TEN lb on HT1080 cells and HT1080 cells transfected with a wild-type TENAR2c gene (HTbetaL.2 cells) were compared using an Alamar Blue™ assay (Biosource #DAL 1100) to measure mitochondrial activity. Cells were plated at subconfluent density in 6-well dishes and were incubated with 0, 50, 500 or 5000 IU/ml of TEN lb for 0, 24 or 48 hours. At the various time points, Alamar Blue reagent was added to cells (1:10 dilution) and incubated on cells for 30 minutes. At 30 minutes, reduced/fluorescent Alamar Blue was detected with fluorescent plate reader. Both HT1080 cells and HTbetaL.2 cells exhibited dose- and time-dependent responses to TEN lb (Figure 3). However, in contrast to HT 1080 cells, HTbetaL.2 cells do not continue to grow from 24 to 48 hours. Although the prohferative rate of HT1080 cells is reduced upon TEN lb treatment, cell number continues to increase. In contrast, HTbetaL.2 cells actually decrease in cell number over time. This decrease is presumably due to apoptotic cell death induced in HTbetaL.2 cells and not in HT1080 cells. A comparison of the anti-growth effects of both TEN and TEN on HT1080 cells transfected with the TENAR2c gene was conducted using a thymidine incorporation assay, described in Example 2 above, using 5000 IU/ml for both human TEN lb (Betaseron) and human TEN 2 (Figure 4). This comparison demonstrated that both of the TENs tested had anti-growth effects on the transfected cells. A comparison of the anti-growth effects of varying concentrations of both human TEN lb and TEN 2 on HT1080 cells transfected with the TENAR2c gene (HTbetaL.2) was also conducted using an Alamar Blue assay (Figure 5). This comparison also demonstrated that TEN had a greater anti-growth effect than TEN , and that this difference in effect between the two TENs was more pronounced at higher concentrations of TEN. Example 4. IFNAR2C transfected HT1080 and MDA231 cells were much more sensitive to the anti-proliferative effects of type I TENs than the parental cell lines. HT1080 cells are epithelial-like cells derived from a human fibrosarcoma. A stable transfected cell line derived from HT1080 (HTbetaL.2), which expressed enhanced levels of TENAR2c, was examined for morphological changes after treatment with TEN lb (Table I). The cells were plated in a 6-well dish and were then treated with TEN lb at a concentration of 500 IU/ml. After two days, untreated cells had formed a confluent carpet of cells (Figure 6, Panel A). TEN lb treated cells had not grown, and less cells were apparent after treatment than before (Figure 6, Panel B). At higher magnifications, the morphology of the treated cells indicated that the cells were undergoing apoptosis (Figure 6, Panel C (20X) and Panel D (40X)). Treated cells appeared to be shedding remnants of cell proteins and DNA in spherical "apoptotic bodies". HT1080 cells were not as severely affected by treatment with TEN lb as HTbetaL.2 cells, and apoptotic bodies were not observed in TEN lb treated HT1080 cells (Figure 6, Panel B). Two stable transfected cell lines derived from MDA231 (MDAbetaL.l and MDAbetaL.2), which expressed enhanced levels of TENAR2c, were examined for 5 morphological changes after treatment with TEN lb (Table I). Cells were treated with TEN lb at a concentration of 500 IU/ml for three days before observation. The growth of parental MDA231 cells was only slightly impaired by treatment with TEN lb, as shown in Figure 7, Panel A (untreated parental cells) and Panel B (treated parental cells). In contrast, MDAbetaL.2 cells were drastically inhibited in their l o growth when treated with 500 IU/ml TEN lb, as shown in Figure 7, Panel C (untreated cells) and Panel D (treated cells). Like HT1080 cells that expressed enhanced levels of TENAR2c, MDAbetaL.2 cells also appeared morphologically to undergo apoptosis when treated with TEN lb. Morphological changes were more difficult to assess in MDA231 cells than in HT1080 cells because normal MDA231 15 cells tend to appear rounded in shape, whereas HT1080 cells normally grow as flat triangles. However, enhanced apoptotic effects were observed in MDAbetaL.2 cells upon treatment with type I TENs. Example 5. HT1080 and MDA231 cells expressing enhanced levels of TENAR2c protein 2 o were examined by TUNEL assay to confirm that the cells undergo apoptosis after treatment with TEN lb. Apoptosis was measured in treated cells using a TdT-mediated dUTP nick end labeling (TUNEL) assay to label fragmented nuclear DNA, which is indicative of apoptosis (In Situ Cell Death Detection Kit, Boehringer Mannheim), hi the TUNEL 25 assay, fluorescent FITC labeled UTP nucleotides were transferred to the ends of fragmented DNA generated that is generated in the nuclei of cells undergoing apoptosis. Apoptotic cells stain bright green under a fluorescent microscope. After four days of TEN treatment, cells in 6-well cell culture plates were fixed for one hour at ambient temperature in IX PBS containing 4% paraformaldehyde. Cells were then 3 o treated for one hour at ambient temperature with 0.3% H2O2 in methanol. Cells were then permeabilized for thirty minutes at ambient temperature in 0.3%Triton-X100 in PBS and were then washed, two times, with PBS. Fixed and permeabilized cells were dried, and cells fields were circled with a PAP pen (Biogenex). Apoptotic nuclei were then enzymatically labeled with the fluorescent label (FITC) as follows. Cells were incubated for forty-five minutes at 37°C with 100 μl staining reagent from Boehringer 5 Mannheim (450 μl Label Solution + 50 μl enzyme solution). Stained cells were washed twice with PBS and were then imaged under a fluorescent microscope. Untreated transfected cells showed a limited number of TUNEL positive nuclei (Figure 8: Panel A, brightfield view; Panel B, fluorescent microscope). Transfected cells that were treated with TEN lb showed a carpet of green TUNEL i o positive nuclei indicating a large fraction of cells were in the final stages of apoptosis (Figure 8: Panel C, brightfield view; Panel D, fluorescent microscope). The small fraction of TUNEL positive cells in untreated cells may reflect endogenous production of low levels of type I TEN. HT1080 parental cells showed absolutely no signs of apoptosis after TEN lb treatment, indicated by a complete lack of TUNEL positive 15 nuclei (Figure 8: Panel E, brightfield view; Panel F, fluorescent microscope). The results obtained with TUNEL assay of MDA231 cells were similar to those obtained with HT1080 cells. Untreated, transfected MDA231 cells (MDAbetaL.2) showed a limited number of TUNEL positive nuclei (Figure 9: Panel A, brightfield view; Panel B, fluorescent microscope). Transfected MDA231 cells 2 o (MDAbetaL.2) that were treated with TEN lb showed a carpet of green TUNEL positive nuclei indicating a large fraction of cells were in the final stages of apoptosis (Figure 9: Panel C, brightfield view; Panel D, fluorescent microscope). Parental MDA231 cells showed only minor signs of apoptosis when treated with 500 IU/ml of TEN lb for four days (Figure 8: Panel C, brightfield view; Panel D, fluorescent 25 microscope). The fraction of TUNEL positive cells in untreated MDAbetaL.2 cells may reflect endogenous production of low levels of type I TEN. Example 6. A human tumor cell line transfected with TENAR2C was more sensitive to the in vivo anti-growth activity of TEN lb than the parental cell line. Injection of LOX human melanoma cells into the tail vein of nude mice will result in the development of defined tumors in the lungs at four weeks post-injection. This model was employed to assess the effectiveness of TEN lb treatment of tumor cells that express enhanced levels of the TENAR2c protein. Nude mice (15 per group) 5 were injected with either parental LOX cells or LOX cells transfected with a gene encoding an IFNAR2c receptor protein. Every other day, starting on day 2 post- injection, mice were injected intraperitoneally with either 200 μg Betaseron or an equivalent volume (200 μl) of saline. At 28 days post-injection, the mice were sacrificed and their lungs were removed and homogenized. DNA was extracted by 0 proteinase K digestion followed by purification over a Qiagen column. The amount of human DNA in the lungs, an indirect measure of the number of LOX parental or LOX-IFNAR2C tumor cells, was quantified by TaqMan PCR, using primers and probes that are specific for the human CCR5 gene. A standard curve was prepared using purified human genomic DNA. 5 Both LOX parental and LOX-IFNAR2c cells were sensitive to the anti-growth activity of systemic Betaseron in vivo, and LOX cells transfected with TENAR2c displayed enhanced sensitivity. Injection of MDA231 cells into the thigh muscle of nude mice will result in the development of defined tumors at two weeks post-injection. This model may be o used to assess the effectiveness of TEN lb treatment of tumor cells that express enhanced levels of the TENAR2c protein. Nude mice are injected with either parental MD A231 cells or MDA231 cells transfected with a gene encoding an TENAR2c receptor protein. The effect of TEN lb administered systemically in inhibiting tumor proliferation in the injected mice is assessed by sacrificing the mice and measuring the 5 size of the tumors. The preceding examples can be repeated with similar success by substituting the generically or specifically described reactants and/or operating conditions of this invention for those used in the preceding examples. From the foregoing description, one skilled in the art can easily ascertain the o essential characteristics of this invention and, without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

Claims: WHAT IS CLAIMED IS: 1. A method of potentiating the effects of a type I TEN on a target cell population comprising increasing the number of functional IFNAR2c receptor chains on the surface of modified cells within the target cell population and then exposing the modified cells to a therapeutically effective amount of a type I TEN. 2. A method according to claim 1, wherein the effects comprise anti-growth effects. 3. A method according to claim 2, wherein the number of functional IFNAR2c receptor chains on the surface of modified cells is increased by up- regulation of gene expression of an IFNAR2c gene. 4. A method according to claim 3, wherein the up-regulation of gene expression of the TENAR2c gene is accomplished by introducing an exogenous gene encoding the IFNAR2c polypeptide into the modified cells. 5. A method according to claim 3, wherein the up-regulation of gene expression of the IFNAR2c gene is accomplished by exposing modified cells of the target cell population to a small molecule which stimulates the promoter of the TJFNAR2c gene. 6. A method according to claim 2, wherein the type I IFN is a type I -IFN, a type I -IFN, a type I -TEN or a consensus type I IFN. 7. A method according to claim 2, wherein the cells of the target cell population are cells involved in a prohferative cell condition. 8. A method according to claim 7, wherein the cells involved in a prohferative cell condition are cancer cells. 9. A method according to claim 7, wherein the cells involved in a prohferative cell condition are smooth muscle cells involved in restenosis. 10. A method according to claim 4, wherein at least one exogenous gene encoding an JJFNAR2c polypeptide is delivered to modified cells using a viral vector. 11. A method according to claim 10, wherein the viral vector is derived from a retrovirus or an adenovirus. 12. A method according to claim 2, wherein the anti-growth effect of the type I IFN on the target cell population is mcreased by at least 5%. 13. A method according to claim 2, wherein the anti-growth effect of the type I IFN is increased by at least 10%. 14. A method according to claim 10, wherein the exogenous gene encoding an TJFNAR2c polypeptide and a gene encoding a type I IFN are delivered to the target cell population as part of the same viral vector. 15. A method potentiating the anti-growth effects of an effector molecule on a target cell population comprising tumor cells, comprising increasing the number of functional effector molecule receptors on the surface of modified cells within the target cell population and then exposing the modified cells to a therapeutically effective amount of a the effector molecule. 16. A method according to claim 15, wherein the effector molecule is a growth factor or an interleukin. 17. A method according to claim 15 wherein the number of effector molecule receptors is increased by the up-regulation of gene expression of a gene encoding the effector molecule receptor. 18. A method according to claim 17, wherein up-regulation of gene expression of the gene encoding the effector molecule receptor is accomplished by the introduction into the modified cells of an exogenous gene encoding the effector molecule receptor. 19. A method according to claim 17, wherein up-regulation of gene expression of the gene encoding the effector molecule receptor is accomplished by exposing modified cells of the target cell population to a small molecule which stimulates the promoter of the gene encoding the effector molecule receptor. 20. A method according to claim 15, wherein the anti-growth effect of the effector molecule on the target cell population is increased by at least 10%. 21. A method of potentiating the effects of a type I IFN on a target cell population comprising increasing the number of functional IFNARl receptor chains on the surface of modified cells within the target cell population and then exposing the modified cells to a therapeutically effective amount of a type I TEN.

Inventors: CROZE ED, RUSSEL-HARDE DEAN, VOGEL DAVID, CROZE, ED, RUSSEL-HARDE, DEAN, VOGEL, DAVID
Applicants: CROZE ED, RUSSEL HARDE DEAN, SCHERING AG, VOGEL DAVID, CROZE, ED, RUSSEL-HARDE, DEAN, SCHERING AKTIENGESELLSCHAFT, VOGEL, DAVID
Application year: 2001
Main classification: A61K48
IPC codes: A61K35, A61K38, A61K45, A61K48, A61P7, A61P9, A61P35, A61P43