FIELD OF THE INVENTION The present invention relates to the treatment of cancer. More particularly, the present invention relates to the treatment of solid tumors which express the Fas receptor by administering to the tumor site or site from which a tumor has been removed non-tumorigenic cells expressing the Fas ligand (FasL).

BACKGROUND OF THE INVENTION One-third of all individuals in the United States will develop some type of cancer during their lifetime. Although the five year survival rate has risen nearly fifty percent as a result of the progress made in early diagnosis and therapy, cancer still remains second only to cardiovascular disease as a cause of death in the United States. Twenty percent of Americans die from cancer, half of which die as a result of lung, breast or colon-rectal cancer.

Cancer is, therefore, a disease that significantly affects the overall health of the human population as a whole.

Despite the great need for effective cancer therapies and the enormous amount of resources employed to develop and optimize such therapies, designing effective treatments for patients with cancer has proven

to be a major challenge. The current regimen of surgical resection, external beam radiation therapy and/or systemic chemotherapy has been partially successful in some kinds of malignancies, but has not produced satisfactory results in others. For example, the above described treatments have proven to be only marginally successful in treating rhabdomyosarcomas, aggressive childhood tumors derived from skeletal muscle, because after treatment, the rhabdomyosarcoma tumors frequently grow back. This is believed to be a result of residual rhabdomyosarcoma tumor cells that were not removed during tumor resection and/or which avoided radiation-induced cell death and/or which gained resistance to the anti-tumor drugs employed for treatment of the tumor. Moreover, the above described treatments exhibit many well known adverse side-effects. There is, therefore, a need for new therapies for effectively treating a variety of malignancies.

One approach to the treatment of cancer has been attempting to induce apoptosis (cell death) of specifically targeted tumor cells and, therefore, molecular mechanisms which function to induce tumor cell apoptosis are of interest. In this regard, the Fas ligand (FasL) protein (also known as CD95L and APO-IL), a cell surface molecule belonging to the tumor necrosis family of proteins, has been shown to induce apoptosis in immune system T-cells that express the Fas receptor. As such, it has been widely reported and believed that the Fas ligand plays an important role in immunoprotection wherein allograft rejection is prevented by Fas ligand- mediated destruction of activated T-cells. Lau et al., Science 273: 109 (1996), Nagata and Golstein, Science 267: 1449 (1995) and Griffith et al., Science 270: 1189 (1995). For example, Lau et al. (1995), supra, report that muscle cells engineered to express the Fas ligand are immunoprotective to transplanted islets of Langerhans, thereby suggesting that the FasL protein plays exclusively an immunoprotective role through the destruction of activated T-cells that express the Fas receptor. We demonstrate herein, however, that contrary to the above described published reports, FasL does not play an immunoprotective role towards cells of solid tumors, but rather

results in FasL-mediated destruction of tumor cells as well as inducing a general immune system response thereagainst.

Seino et al., Nature Medicine 3 (2): 165 (1997) demonstrate that tumor cells engineered to express the Fas ligand were capable of inducing the rejection of Fas receptor-negative tumor cells in vivo, primarily by a neutrophil-mediated response, however, no in vitro effects were observed.

However, Seino et al. employ tumor cells as the source of Fas ligand, which carries with it numerous undesirable attributes.

Therefore, it is desirable to provide a method of treating solid tumors where FasL is administered to the tumor site without the requirement of administering tumorigenic cells to that site.

It is also desirable to provide a method of treating solid tumors where the administration of FasL induces apoptosis of tumor cells which express the Fas receptor but where the administered non-tumorigenic cells do not self-destruct because they do not express the Fas receptor.

It is also desirable to provide a method of treating rhabdomyosarcoma tumors by implanting myoblasts which do not express the Fas receptor, but which do express the Fas ligand, at the tumor site or at the site from which a tumor has been removed by surgical resection, radiation treatment and/or chemotherapy.

In addition, it is desirable to implant FasL-expressing myoblasts at the site of a tumor or at the site from which a tumor has been removed to produce a soluble form of FasL that will travel through areas inaccessible to the myoblasts themselves, such as between cells of a layer of cells.

It is also desirable to implant FasL-expressing cells at the site of a tumor or at the site from which a tumor has been removed in conjunction with other known cancer therapies.

It is also desirable to induce the movement of neutrophils and other immune system cells, such as T-cells, to the site of a tumor or the site from which a tumor has been removed to stimulate a more general immune response against tumor cells present at that site; a response commonly known as a bystander effect.

It is also desirable to provide an in vitro assay based upon the ability of FasL expressing cells to induce destruction of Fas receptor expressing cells for determining whether a particular cell expresses the Fas receptor and, therefore, is a candidate for FasL-mediated killing with the presently described method.

These and further objects will be apparent to the ordinarily skilled artisan upon consideration of the specification as a whole.

SUMMARY OF THE INVENTION In accordance with the present invention, a method for the treatment of solid tumors which express the Fas receptor is provided. The presently described method comprises administering FasL to the site of a tumor or the site from which a tumor has been removed. FasL may also be administered to tumor cells in vitro in an assay to determine diagnostically if those tumor cells express the Fas receptor. In a particularly preferred embodiment of the present invention, Fas receptor-negative myoblasts which are genetically engineered to express soluble or membrane-bound FasL are implanted into a patient at the site of a tumor or the site from which a tumor has been removed, thereby inducing Fas ligand-mediated apoptosis of Fas receptor expressing tumor cells. In one embodiment, the Fas receptor expressing tumor cells which are killed are rhabdomyosarcoma cells.

The results described herein show that the claimed method of treating cancerous tumors results in the destruction of neighboring Fas receptor expressing tumor cells by membrane-bound and soluble FasL, in addition to a general immune provocation that stimulates the clearance of the tumor cells. This general immune provocation involves not only the infiltration of neutrophils into the area of administration of FasL-expressing cells, but also involves the infiltration of T-cells as well. The presently described method is advantageous in that a preferred embodiment utilizes primary, non-tumorigenic cells to provide a system where the cells are easily purified and are readily recombinantly transduced with the gene expressing

FasL. In a particularly preferred embodiment, the non-tumorigenic cells engineered to express the Fas ligand are primary myoblasts.

In yet another preferred embodiment, the Fas receptor-negative cells which are engineered to express FasL are obtained from human patients having autoimmune lymphoproliferative syndrome (ALPS) (Fisher et al., Ce// 81: 935 (1995)), a disease characterized by a mutation in the Fas receptor, such that those cells are effectively Fas receptor-negative. ALPS is the human equivalent to the Ipr mutation in the mouse. As such, there exists a supply of Fas receptor-negative universal donor cells which may be transduced with the gene encoding Fas ligand and these cells will not self- destruct when expressing the Fas ligand.

Additionally, one may exploit the allogenic nature of various donor cells in order to elicit a general immune response to the site of implantation, thereby further enhancing the ability to destroy tumor cells at that site.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 A depicts a map of the retroviral construct used to transduce primary skeletal myoblasts isolated from C3H mice and Ipr mice, thereby allowing those cells to express FasL. The map represents a pBABE M derived retrovirus with an LTR promoter containing the FasL coding sequence (FasL) followed by an internal ribosome entry sequence (IRES) and a Green Fluorescent Protein (GFP) reporter gene. Figure 1 B depicts three graphs showing Fluorescence Activated Cell Sorting (FACS) analyses where the following cell types are shown: untransduced myoblasts as a control (top), C3H (wild type) harboring the construct shown in Fig. 1 A (middle) and Ipr (mutant, Fas receptor defective) harboring the construct shown in Fig. 1 A (bottom).

Figure 2A depicts a bar graph showing the induction of apoptosis in Jurkat T cells by FasL-expressing myoblasts. Specifically, Jurkat T cells, which express Fas receptor and are therefore sensitive to FasL-mediated killing, were co-cultured with C3H myoblasts engineered to express FasL (C3H FasL), Ipr myoblasts engineered to express FasL (isolated from Fas

receptor-deficient B6-lpr mice; Ipr FasL) or non-transduced Ipr cells as a control (Ipr). Figure 2B depicts a bar graph showing the results obtained with a cell culture supernatant from Ipr myoblasts engineered to express FasL. Killing of Jurkat T cells is assessed as a percentage of total cells stained by ethidium bromide after 4 hours.

Figure 3 depicts photographs of C3H cells at 24 (left top) or 48 hours (left bottom) in low-serum growth medium, and C3H cells engineered to express FasL at 24 (right top) or 48 hours (right bottom) in low serum growth medium showing self-destruction due to expression of both Fas receptor and FasL upon myoblast fusion.

Figure 4 depicts graphs obtained from FACS analysis of either BNL cells, liver-derived cells known to express the Fas receptor (top), Ipr cells (middle) or C3H cells (bottom) stained with an antibody against Fas receptor (Jo-2) coupled to the fluorescent molecule phycoerythrin (PE). The darker lines represent fluorescence detected after staining with the Jo-2 antibody whereas the lighter lines represent background detection without antibody staining. These results show the reduction of Fas receptor on mutant Ipr myoblasts.

Figure 5 depicts a bar graph showing the target cell death in percentage of different cell types cultured with either Ipr cells (Ipr), Ipr cells engineered to express FasL (Ipr FasL) or anti-human Fas receptor antibody (anti hFas AB). From left to right the following are shown: Mb50 normal human myoblasts (control); and the following four well characterized rhabdomyosarcoma lines which were isolated from four different individuals, Rh1, Rh18, Rh28 and Rh30. (Houghton et al., Cancer Chemotherapy and Pharmacology 33 : 265-272 (1994) and Shapiro et al., Cancer Res. 50: 6002- 6009 (1990)).

Figure 6 depicts graphs of a FACS analysis showing from top to bottom the human myoblast and rhabdomyosarcoma cell lines described in Figure 5, Mb50 (control), Rh1, Rh18, Rh28 and Rh30, where the cell lines were stained with a PE-labeled antibody to human Fas receptor, Jo-2. The darker lines represent fluorescence detected after staining with the Jo-2

antibody whereas the lighter lines represent background detection without antibody staining. The results demonstrate that all of the tested lines except for Rh30 express the Fas receptor.

Figure 7A depicts a photograph of a low power magnification of a kidney at day three after untransduced Ipr myoblasts have been implanted under the kidney capsule. The section is stained with hematoxylin and eosin (H&E). Arrows point to the approximate site of implantation. Figure 7B depicts a photograph of a low power magnification of a kidney at day three after Ipr myoblasts transduced with the FasL gene have been implanted under the kidney capsule. The section is stained with H&E. Arrows point to the approximate site of implantation where inflammation is clearly evident.

Figure 7C depicts a photograph of a high power magnification of the kidney shown in Figure 7A which is stained with H&E. A lack of neutrophil infiltration is evident. Figure 7D depicts a photograph of a high power magnification of the kidney shown in Figure 7B which is stained with H&E.

A massive infiltration of neutrophils under the kidney capsule is evident.

Figure 7E depicts a photograph of a high power magnification of a kidney at day seven after untransduced Ipr myoblasts were implanted under the kidney capsule. The kidney has been immunostained with an antibody directed against desmin, which only stains the implanted myoblasts brown, and also counterstained to visualize nuclei which appear light blue. It is noted that several multinucleate, differentiated myotubes can be seen under the kidney capsule. Figure 7F depicts a photograph of a high power magnification of a kidney at day seven after Ipr myoblasts transduced with the FasL gene were implanted under the kidney capsule. The kidney has been immunostained with an antibody directed against desmin, which only stains the implanted myoblasts brown, and also counterstained to visualize nuclei which appear light blue. It is noted that the implanted myoblasts are being engulfed by the neutrophil infiltration.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides materials and methods useful for killing cells that express the Fas receptor protein on their cell surfaces. In accordance with the present invention, we herein describe for the first time that non-tumorigenic celrs which have been genetically engineered to recombinantly express either a membrane-bound or soluble form of the Fas ligand protein are capable of mediating the destruction of cells that express the Fas receptor protein. Therefore, the present invention provides a useful method for therapeutical treating cancerous tumors comprising tumor cells that express the Fas receptor protein.

As used herein, the terms"Fas ligand","FasL", and grammatical equivalents thereof (also known as CD95L and APO-IL), refer to membrane- associated and soluble polypeptides that are capable of inducing apoptosis of cells expressing the Fas receptor. The nucleic acid and amino acid sequences of the Fas ligand have been previously described by Watanabe- Fukunaga, et al., J. Immunology 148: 1274 (1992). The FasL nucleic acid and amino acid sequences are shown in Figure 1 of the Watanabe-Fukunaga article referenced above and are set forth herein as SEQ ID NO : 1 and SEQ ID NO : 2, respectively.

As used herein, the terms Fas ligand or equivalents thereof are also' intended to encompass polypeptides encoded by nucleic acids which hybridize under high stringency conditions to nucleic acids having the sequence of SEQ ID NO : 1 and which are capable of inducing apoptosis of cells expressing, the Fas receptor. Convenient techniques for preparing variants of the Fas ligand protein of SEQ ID NO : 2 (for example, Fas ligand variants that possess one or more conservative amino acid substitutions from the amino acid sequence of SEQ ID NO : 2) are well known in the art and may routinely be employed herein. Such techniques include, for example, well known site-directed mutagenesis techniques. Additionally, convenient techniques for screening FasL variant polypeptides for apoptosis-inducing activity are described below and are routine in nature.

As used herein, the term"Fas"refers to the Fas receptor or any fragment thereof capable of binding to FasL as described in Larsen et al., Transplantation 60: 221 (1995). In a particularly preferred embodiment, the Fas receptor is the membrane-bound Fas receptor polypeptide that is naturally expressed on the surface of a variety of different mammalian cells.

Tumor cells which are susceptible to FasL-mediated cell killing by the method of the present invention include all tumor cells which express the Fas receptor. Such tumor cells may be routinely identified by reaction with detectably-labeled anti-Fas receptor antibodies, such as antibody Jo-2 which is commercially available from Pharmingen, San Diego, California or by co- culture with non-tumorigenic cell lines which express the Fas ligand.

Examples of tumor cells which naturally express the Fas receptor include, but are not limited to, cells derived from virtually all solid tumors such as rhabdomyosarcoma-derived cells and cells derived from pancreatic, breast and lung tumors and especially those solid tumors which are not easily accessible to surgical resection.

The phrase"site from which a tumor has been removed"refers generally to the site where a tumor has been removed, for the most part by surgical excision, radiation and/or chemotherapy. This site generally has some lingering tumor cells in the area which are being targeted by the method described herein.

"Allogeneic cells"are cells derived from a genetically distinct individual of the same species. When an animal is immunized with allogeneic cells, it elicits the infiltration of cytotoxic T lymphocytes specific for the major histocompatibility complex (MHC) molecules on the allogeneic cells.

Moreover, other immune responses such as infiltration of neutrophils may occur.

"Neutrophil infiltration"refers to the neutrophilic chemotaxis which is induced by the administration of allogenic FasL expressing cells in vivo.

"Genetically engineered cells"means cells that do not naturally express the polypeptide product of interest. However, due to genetic engineering, such as transforming or transducing the cells with a gene encoding the polypeptide product of interest, the cells express the polypeptide product of interest.

"Expression system"or"construct"refers to nucleic acid sequences containing a desired coding sequence and control sequences in operable linkage, so that hosts transformed or transduced with these sequences are capable of producing the encoded proteins. To effect transformation, the expression system may be included on a vector; however, the relevant DNA may then also be integrated into the host chromosome.

As used herein,"cell,""cell line,"and"cell culture"are used interchangeably and all such designations include progeny. Thus, "transformants"or"transformed or transduced cells"includes the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny may not be precisely identical in nucleic acid content, due to deliberate or inadvertent mutations.

Mutant progeny that have the same functionality as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.

"Plasmids"are designated by a lower case p preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are commercially available, are publicly available on an unrestricted basis, or can be constructed from such available plasmids in accord with published procedures. In addition, other equivalent plasmids are known in the art and will be apparent to the ordinary artisan.

"Transformation"or"transduction"means introducing nucleic acids into an organism so that the nucleic acids are replicable, either as an extrachromosomal element or by chromosomal integration. Depending on the host cell used, transformation is done using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride,

as described by Cohen, Proc. Natl. Acad. Sci. USA 69,2110 (1972) and Mandel et al., J. Mol. Biol. 53,154 (1970), is generally used for prokaryotes or other cells that contain substantial cell-wall barriers. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology 52,456 (1978) is preferred. General aspects of mammalian cell host system transformations have been described by Axel in U. S. Pat. No. 4,399,216 issued August 16,1983. However, other methods for introducing nucleic acids into cells such as by nuclear injection, electroporation, lipofection or by protoplast fusion may also be used.

"Retroviral infection"of host cells with nucleic acid encoding FasL may be performed as follows. The primary host cell line is grown in F- 10/DME medium, 20% fetal bovine serum (FBS), 2.5 ng/ml basic fibroblast growth factor (bFGF), in either a 6-well or 6 cm collagen-coated dish at 5% CO2. Cells should generally not be seeded at less than about 10% confluence. An aliquot of the growth medium may then be removed, and replaced with undiluted, unconcentrated viral supernatant supplemented with 8, ug/ml polybrene. The viral supernatant is supplemented to a final concentration of 20% FBS and 2.5 ng/ml bFGF. The culture dish is then returned to the incubator for approximately 15 minutes, wrapped in parafilm and spun in a microplate carrier in a tabletop centrifuge at 32°C for approximately 30 minutes. The growth medium is then replaced with fresh medium and the plate returned to the incubator. The above procedure may then be repeated with new viral supernatant approximately every 8 hour.

Hybridization of nucleic acids is preferably performed under "stringent conditions"which means (1) employing low ionic strength and high temperature for washing, for example, 0.015 sodium chloride/0. 0015 M sodium citrate/0.1 % sodium dodecyl sulfate at 50°C, or (2) employing during hybridization a denaturing agent, such as formamide, for example, 50% (vol/vol) formamide with 0.1 % bovine serum albumin/0. 1 % Ficoll/0. 1 % polyvinylpyrrolidone/50 nM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C. Another example is use of

50% formamide, 5 x SSC (0.75 M NaCI, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6/8), 0. 1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50, ug/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC and 0.1 % SDS.

Yet another example is hybridization using a buffer of 10% dextran sulfate, 2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C. When a nucleic acid sequence of a nucleic acid molecule is provided, other nucleic acid molecules hybridizing thereto under the conditions described above are considered within the scope of the sequence.

"Non-tumorigenic cells"are cells that are not immediately capable of developing into a tumor.

General Methodologies : General methodologies for muscle cell-mediated gene therapy have been previously described. Blau and Springer, New England J. Med., 333: 1554 (1995). Generally, a gene encoding a polypeptide therapeutic, in this case, FasL as shown in SEQ ID NO : 1, is cloned into a retroviral vector.

Preferred retroviral vectors include, for example, MFG and pBABE, however, other non-retroviral vectors, such as adeno-, adeno-associated and lenti- viruses will find use herein. Primary myoblasts may be isolated from muscle, in this case from Fas receptor-deficient skeletal muscle cells. Techniques for isolating myoblasts are described in Rando and Blau, J. Cell. Biol.

125 (6): 1275 (1994). However, it is understood that rather than using Fas receptor deficient cells, cells that have been genetically engineered to not express the Fas receptor can also be used. The non-tumorigenic cells of choice are infected with retrovirus, or the FasL gene is incorporated into the cells by any other convenient means, and the population increased. Non- tumorigenic cells expressing recombinant FasL protein are then injected or otherwise administered into the in vivo site of choice or are employed in vitro. The only limitation on the non-tumorigenic cells which find use in the present invention is that they express the Fas ligand, either naturally or

recombinantly, and that they preferably do not express the Fas receptor, although cells that do express the Fas receptor may also find use. Many of such cells are known in the art and will find use in the present invention.

Preferably, the non-tumorigenic cells employed are myoblast cells, however, other cells such as fibroblasts, keratinocytes, and the like may also be employed. Often, the non-tumorigenic cell of choice will depend upon the site at which the tumor to be treated exists, wherein non-tumorigenic cells that are of a similar derivation as those cells surrounding the site of the tumor are preferred.

The in vivo administration of living cells has been previously described and is well known in the art. Gussoni et al., Nature 356: 435 (1992), Pavlath et al., J. Cell Biol. 127: 1923 (1994) and Rando et al., Exp.

Cell Research 220 : 383 (1995). The Fas ligand-expressing cells described herein may be administered in vivo using these well known techniques, wherein the number of cells per administration may be determined empirically in a routine manner. Generally, in a preferred method, 5X106 cells are implanted to the site where a tumor has been excised immediately after the tumor has been excised. Alternatively, the cells can be administered independently of other methods of treatment, i. e., at an unmodified tumor site. The number of cells administered and the method of administration may differ depending upon the desired application, however, appropriate treatment programs may routinely be prepared by those skilled in the art.

The FasL-expressing non-tumorigenic cells may be administered in combination with any number of known carrier compounds including, for example, phosphate buffered saline, or other appropriate buffered medium, and the like. Additionally, the Fas ligand-expressing cells may administered in conjunction with the administration of one or more other agents that have tumor cell killing capability. Therefore, the Fas ligand-expressing cells described herein may be administered in conjunction with any of a number of known and routinely employed chemotherapeutic agents. Additionally, in a preferred embodiment, Fas ligand-expressing cells may be administered in combination with the administration of the ONYX-015 mutant adenovirus

that has been shown to be capable of selectively infecting and killing cells that do not express the p53 tumor suppressor protein (Bischoff et al., Science 274: 373 (1996)).

The following examples are meant to be illustrative and in no way should be used to limit the scope of the claims.

Specific Examples : General overview Nucleic acid encoding the Fas ligand was introduced by retroviral transduction to myoblasts to determine whether these cells would effectively recombinantly express FasL. It was then to be verified, contrary to previous reports, whether the self-destruction of myoblasts was mediated by the Fas pathway. To determine whether the myoblasts express the Fas receptor, the myoblasts of two strains of wild-type mice were assayed both by reverse transcriptase polymerase chain reactions (RT-PCR) and by cell sorting with a Fas receptor-specific antibody. It was then to be determined whether myoblasts isolated from the Ipr mouse, which harbors a genetic defect in the Fas receptor, did not express Fas receptor in either assay or undergo apoptosis. It was then to be determined whether FasL expressing Ipr myoblasts had the ability to kill human rhabdomyosarcomas isolated from patients.

Overexpressionof FasL in primary myoblasts Nucleic acid encoding the Fas ligand was introduced into primary myoblasts by retroviral transduction and shown to be expressed by greater than 95% of myoblasts by cell sorting. Specifically, myoblasts derived from the wild-type mouse strain C3H and the Fas receptor-deficient mutant strain Ipr were transduced with the construct shown in the diagram of Figure 1 A.

The construct is formed of a pBABE M derived retrovirus containing the FasL coding sequence (FasL) followed by an internal ribosome entry sequence (IRES) and a Green Fluorescent Protein (GFP) reporter gene. In this construct, the viral promoter drives the expression of a bicistronic RNA

coding for FasL and an IRES controlled GFP. The presence of the GFP allows determination of the efficiency of transduction of C3H and Ipr myoblasts obtained with this construct.

All cloning steps were performed as previously described (Sambrook et al.,"Molecular Cloning : A Laboratory Manual"2nd ed., Cold Spring Harbor Laboratory Press, 1989). Mouse FasL cDNA was isolated from total testis RNA by RT-PCR and subsequently sequenced. FasL specific primers were designed to introduce a Ncol restriction endonuclease site at the start codon (3'-GCGCCATGGGCCAGCAGCCCATGAATT ACCCATGTCCCCA-5') (SEQ ID NO : 3) and a BamHl restriction endonuclease site at the 3'end (5'-GCGGGATCCTTAAAGCTTATACAAGCCG'3') (SEQ ID NO : 4) of the cDNA for convenient cloning. FasL cDNA was then cloned into a pBABE M vector. A unit consisting of the green fluorescent protein (GFP) cloned downstream of a mengo derived internal ribosome entry site (IRES) was cloned downstream of the FasL cDNA in pBABE M to generate the retroviral vector pBABE M FasL-IRES-GFP as depicted in Figure 1A.

The vector was introduced into the test cells and Fluorescence Activated Cell Sorting (FACS) on those cells was then performed as follows.

Cells analyzed for GFP expression were trypsinized, rinsed in phosphate buffered saline (PBS) and resuspended in FACS buffer (PBS, 4% fetal bovine serum [FBS] and propidium iodine) for the FACS analysis. The cells were then analyzed by FACS.

Figure 1 B shows the Fluorescence Activated Cell Sorting (FACS) analysis of the above described cells. As shown in Figure 1 B, FACS analysis of the cell lines revealed that greater than 95% of the transduced cells expressed the GFP reporter, thereby indicating the expression of the construct, and specifically FasL, in those cells.

Induction of apoptosis by FasL-expressing primary myoblasts In order to confirm whether this population of transduced myoblasts expressed a biologically active Fas ligand, in vitro cell killing assays were performed. As such, Jurkat T cells, a cell line known to express the Fas receptor and which are therefore sensitive to FasL-mediated apoptosis, were co-cultured for 4 hours with Ipr myoblasts (Ipr) as a control or C3H myoblasts (C3H FasL) or Ipr (Ipr FasL) myoblasts which have been engineered as described above to express the FasL gene product. As shown in Figure 2A, both C3H and Ipr myoblasts expressing FasL were able to induce significant apoptosis in the Fas receptor-expressing Jurkat cell line whereas the Ipr cells which do not express FasL are incapable of inducing apoptosis of Fas receptor-expressing Jurkat cells. Notable about the results is that the extent of apoptosis in the target Jurkat cells increased with an increasing ratio of myoblasts to Jurkat cells (data not shown).

As shown in Figure 2B, a 100,000xg cell culture supernatant from the FasL-expressing Ipr myoblasts was also able to induce apoptosis in Jurkat T cells, even if cellular debris was removed. These results, therefore, demonstrate that a soluble form of FasL is released by the cells and that this soluble form of FasL is capable of killing Fas receptor expressing cells. This activity could never be observed with supernatants obtained from untransduced control myoblasts (data not shown).

Apoptotic suicide of myoblast cultures expressing FasL Primary C3H myoblasts do not express the Fas receptor and, therefore, transduction of those cells with a FasL expression vector did not result in significant killing of the cells when grown in standard growth medium containing 20% FBS (data not shown). However, normal C3H and C3H FasL-expressing myoblasts were then induced to differentiate by culturing the cells in low-serum culture medium (DMEM containing 2% horse serum). As shown in Figure 3, by 24 hours the cells were beginning to differentiate and were indistinguishable. By 48 hours, the cells not expressing FasL had fused into myotubes, while many of the cells expressing

FasL were dead. By 72 hours, all of the FasL expressing myoblasts were dead. In contrast, FasL-expressing Ipr myoblasts, which are defective in the Fas receptor, did not die even after 15 days in differentiation medium (not shown).

Remarkably arid unexpectedly, as shown in Figure 3, Fas ligand- expressing myoblasts underwent apoptosis in tissue culture to a far greater extent than that shown by Walsh et al., Science 273: 359 (1996) in the course of normal muscle differentiation. Moreover, contrary to the results presented by Lau et al., Science 273: 109 (1996), the FasL expressing myoblasts self-destruct.

Confirmation that myoblasts express Fas receptors Various cells were next analyzed for Fas receptor expression as follows. Cells analyzed for Fas receptor expression were rinsed with PBS, and incubated for 2 to 20 minutes in PBS containing 1 mM EDTA in order to detach the cells from the culture dish. The cell suspension (2x105 cells) was rinsed twice in PBS and incubated for 20 minutes on ice in staining solution containing deficient RPMI, 4% FBS and HEPES containing 0.5, ug/ml of the phycoerythrin (PE)-labeled Jo-2 antibody (Pharmingen, San Diego, California).

Human cells were incubated according to the manufacturers recommendations. After the staining procedure, cells were rinsed with staining buffer and analyzed by FACS.

Using the above methodology, BNL cells, a liver-derived cell line known to express the Fas receptor, Ipr and C3H myoblast cells were stained with approximately 1, ug/ml of PE-labeled Jo-2 antibody. The stained cells where then analyzed by FACS and the results are shown in Figure 4. As shown in Figure 4, the BNL liver-derived cell line exhibited a significant increase in fluorescence when stained with the Jo-2 antibody (dark line) over the baseline levels without antibody (light line). Similarly, C3H myoblasts exhibited an increase in fluorescence in the presence of the antibody, confirming that these cells also express the Fas receptor. In contrast, however, Ipr myoblasts, which are known to be deficient in the Fas receptor,

did not exhibit a significant increase in fluorescence. These results verify that the cell death observed in C3H myoblast cultures overexpressing FasL was a result of Fas-mediated auto-destruction.

In this regard, that the auto-destruction of myoblasts was mediated by the Fas pathway was verified by showing that, contrary to previous reports, the myoblasts of two strains of wild-type mice express Fas receptor as assayed both by RT-PCR and by cell sorting with a murine receptor- specific antibody (Figure 4). By contrast, myoblasts isolated from the Ipr mouse, which harbors a genetic defect in the Fas receptor, did not express Fas receptor in either assay or undergo apoptosis. Moreover, when Ipr cells expressing Fas ligand were implanted into the kidney capsule, immunoprotection was not observed in the presence or absence of islets of Langerhans cells, thereby contradicting the teachings of Lau et al., Science 273: 109 (1996) (see below). Indeed, to the contrary, the Fas ligand expressing cells served as chemoattractants for neutrophils and the rejection of myoblasts with or without islets was significantly faster than that observed with controls.

Killing of rhabdomyosarcomas by co-culture with FasL-expressincr myoblasts We next tested for the ability of FasL-expressing cells to kill human rhabdomyosarcomas isolated from patients in vitro. Specifically, samples of four rhabdomyosarcomas isolated from four different human patients as well as the human myoblast line Mb50 were individually co-cultured with Ipr myoblasts expressing (Ipr FasL) or not expressing (Ipr) FasL or with an anti- human Fas receptor antibody (anti hFas AB). The rhabdomyosarcoma- derived cells tested are the well characterized lines designated Rh1, Rh18, Rh28 and Rh30 (Houghton et al., (1994) supra and Shapiro et al., (1990) supra). Each showed significant killing in the presence of Fas ligand- expressing Ipr myoblasts as compared to untransduced myoblasts, although two (Rh1 and Rh18) showed a significantly greater percentage of killing than the other lines tested. Cells cultured with an anti-human Fas receptor

antibody showed no significant level of cell death. Thus, the Fas ligand was far more potent than the antibody in the killing assays.

The above results demonstrate the ability of Fas ligand-expressing Ipr myoblasts to kill human rhabdomyosarcomas isolated from patients. Cell sorting revealed that as many as 55% of rhabdomyosarcomas were destroyed upon c-culture with Fas ligand-expressing Ipr myoblasts after only 24 hours (Figure 5).

Confirmation that rhabdomvosarcomas ex, press Fas The samples of four rhabdomyosarcomas, Rh1, Rh18, Rh28 and Rh30 shown in Figure 5 were then stained with an PE-labeled antibody to Fas receptor, Jo-2, and sorted by FACS as done in Figure 4. As shown in Figure 6, the MB50 myoblasts were positive for Fas receptor expression as predicted by the results in Figure 5. Three out of four of the rhabdomyosarcomas also exhibited detectable Fas receptor in this assay (Rh1, Rh18 and Rh28) whereas only Rh30 tested negative for the presence of Fas receptor. Two of the four rhabdomyosarcoma lines were effectively killed by FasL expressing Ipr cells. The other two rhabdomyosarcoma lines presumably have an alteration in the signal transduction pathway downstream of Fas. In any event, unlike previous in vitro studies with cancer cells, these studies provide evidence for a direct killing effect of Fas ligand on tumor cells in vitro and suggest that the present Fas receptor/FasL system is similar to that where the presence of estrogen receptor is an indicator of whether tamoxifen will be effective in the treatment of breast cancer. Thus, the presence of Fas receptors in tumor cells serves as an excellent indicator of whether those cells are susceptible to Fas ligand- mediated cell destruction.

These results further demonstrate the ability of Fas-ligand expressing Ipr myoblasts to kill human rhabdomyosarcomas isolated from patients. That cell sorting revealed that in 3 of the 4 cases tested, the extent of apoptosis was correlated with the expression of Fas receptor, taken together with the findings shown in Figure 5, indicate that mutant Fas

receptor negative myoblasts genetically engineered to express the Fas ligand are prevented from self-destruction. Moreover, they can effectively kill Fas receptor expressing muscle-derived tumor cells.

As an alternative to the Ipr cells, human cells obtained from persons inflicted with autoimmune lymphoproliferative syndrome (ALPS), the human counterpart to the Ipr mutation in the mouse, provide an excellent source for universal donor human cells which lack the Fas receptor and which could be engineered to express the Fas ligand for use in the present invention. Obtaining primary cells from a tissue sample taken from such an individual can be performed using well known techniques (e. g., see Rando and Blau, (1994) supra. Overall, the above described method allows a three- pronged attack on this devastating human malignancy of children via direct Fas ligand-mediated killing and immunoattraction, both by neutrophils and T- cells.

While a minimal amount of endogenous myoblasts may be killed locally by the general immune response directed to the site of administration, this is not a problem since regeneration of muscle is very efficient.

Moreover, the potential loss of some normal tissue will be more than compensated for by the eradication or reduction of the cancer.

Myoblasts expressing FasL elicit rapid neutrophilic infiltration The effects of the administration of FasL-expressing myoblasts in vivo was determined as follows. To avoid Fas receptor/FasL mediated suicide of administered myoblasts, primary myoblasts obtained from Fas receptor-deficient B6-lpr mice were generated. FasL-transduced Ipr myoblasts differentiated normally in vitro. Non-transduced or FasL transduced Ipr myoblasts were administered by injection under the kidney capsule of congenic B6 mice (2 x 106 myoblasts/kidney, 10 mice/group).

Mice were sacrificed at days 1,3,7,14, and 26 days post transplant. The gross appearance of kidneys taken at various time points is shown in Figure 7.

As shown in Figure 7A, 7C and 7E, kidneys to which non- transduced Ipr myoblasts were administered appeared normal at all time points, except for a subtly darker area containing the myoblasts. In contrast, the kidneys transplanted with FasL expressing Ipr myoblasts had raised white lesions resembling an abscess, which appeared by day 1 and disappeared by day 26. On histological examination, FasL-transduced Ipr myoblasts were found to provoke an intense neutrophilic infiltrate and appeared completely destroyed within 7 days (Figure 7B, 7D and 7F). FasL-transduced myoblasts transplanted into Fas-deficient Ipr hosts also persisted over time, demonstrating a requirement for host Fas receptor expression for the granulocytic response (data not shown). Therefore, these results demonstrate that the in vivo administration of cells expressing the Fas ligand functions to induce apoptosis of tumor cells expressing the Fas receptor.

Myoblasts expressing FasL are capable of inhibiting the formation of rhabdomyosarcoma tumors in immunodeficient mice To determine if myoblasts that express FasL are capable of inhibiting the formation of rhabdomyosarcoma tumors in immunodeficient mice, the following experiments were performed. Cells derived from a human rhabdomyosarcoma (Rh) tumor were implanted into the leg muscles of imrnunodeficient mice either in association with myoblasts that do not express the Fas ligand or myoblasts that had been recombinantly engineered to express the Fas ligand protein. When Rh cells were implanted into the leg muscles of mice in combination with myoblasts that do not express the Fas ligand polypeptide, large tumors formed within three weeks that were visible by histological staining. In contrast, however, when Rh cells were implanted into the leg muscles of mice in combination with myoblasts that were engineered to express the Fas ligand polypeptide, histological examination of the injected leg muscles revealed that tumors were absent in every case. A small region of inflammation containing many neutrophils was observed at the injection site. The absence of human tumor cells in the mouse muscle

was confirmed by staining tissue sections with Hoechst 33258 dye, which stains human nuclei uniformly and stains mouse nuclei in punctate fashion.

The foregoing description details specific methods which can be employed to practice the present invention. Having detailed such specific methods, those skilled in the art will well enough known how to devise alternative reliable methods at arriving at the same information in using the fruits of the present invention. Thus, however, detailed the foregoing may appear in text, it should not be construed as limiting the overall scope thereof; rather, the ambit of the present invention is to be determined only by the lawful construction of the appended claims. All documents cited herein are expressly incorporated by reference.

SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Blau, Helen Hofmann, Andreas (ii) TITLE OF INVENTION : TREATMENT OF CANCER BY THE ADMINISTRATION OF FAS LIGAND EXPRESSING NON-TUMORIGENIC CELLS (iii) NUMBER OF SEQUENCES: 4 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Flehr, Hohbach, Test, Albritton & Herbert (B) STREET: Four Embarcadero Center, Suite 3400 (C) CITY: San Francisco (D) STATE: California (E) COUNTRY: United States (F) ZIP: 94111-4187 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: PatentIn Release #1. 0, Version #1. 30 (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US Unknown (B) FILING DATE: Herewith (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Brezner, David J.

(B) REGISTRATION NUMBER: 24,774 (C) REFERENCE/DOCKET NUMBER: A-64601/DJB/MTK (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (415) 781-1989 (B) TELEFAX: (415) 398-3249 (C) TELEX: 910 277299 (2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1506 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 50.. 1030 (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GCCGCAGGCT GCCCACACAG GCCGCCCGCT GTTTTCCCTT GCTGCAGAC ATG CTG 55 Met Leu 1

TGG ATC TGG GCT GTC CTG CCT CTG GTG CTT GCT GGC TCA CAG TTA AGA 103 Trp Ile Trp Ala Val Leu Pro Leu Val Leu Ala Gly Ser Gln Leu Arg 5 10 15 GTT CAT ACT CAA GGT AGT AAT AGC ATC TCC GAG AGT TTA AAG CTG AGG 151 Val His Thr Gln Gly Ser Asn Ser Ile Ser Glu Ser Leu Lys Leu Arg 20 25 30 AGG CGG GTT CAT GAA ACT-GAT AAA AAC TGC TCA GAA GGA TTA TAT CAA 199 Arg Arg Val His Glu Thr Asp Lys Asn Cys Ser Glu Gly Leu Tyr Gln 35 40 45 50 GGA GGC CCA TTT TGC TGT CAA CCA TGC CAA CCT GGT AAA AAA AAA GTT 247 Gly Gly Pro Phe Cys Cys Gln Pro Cys Gln Pro Gly Lys Lys Lys Val 55 60 65 GAG GAC TGC AAA ATG AAT GGG GGT ACA CCA ACC TGT GCC CCA TGC ACA 295 Glu Asp Cys Lys Met Asn Gly Gly Thr Pro Thr Cys Ala Pro Cys Thr 70 75 80 GAA GGG AAG GAG TAC ATG GAC AAG AAC CAT TAT GCT GAT AAA TGC AGA 343 Glu Gly Lys Glu Tyr Met Asp Lys Asn His Tyr Ala Asp Lys Cys Arg 85 90 95 AGA TGC ACA CTC TGC GAT GAA GAG CAT GGT TTA GAA GTG GAA ACA AAC 391 Arg Cys Thr Leu Cys Asp Glu Glu His Gly Leu Glu Val Glu Thr Asn 100 105 110 TGC ACC CTG ACC CAG AAT ACC AAG TGC AAG TGC AAA CCA GAC TTC TAC 439 Cys Thr Leu Thr Gln Asn Thr Lys Cys Lys Cys Lys Pro Asp Phe Tyr 115 120 125 130 TGC GAT TCT CCT GGC TGT GAA CAC TGT GTT CGC TGC GCC TCG TGT GAA 487 Cys Asp Ser Pro Gly Cys Glu His Cys Val Arg Cys Ala Ser Cys Glu 135 140 145 CAT GGA ACC CTT GAG CCA TGC ACA GCA ACC AGC AAT ACA AAC TGC AGG 535 His Gly Thr Leu Glu Pro Cys Thr Ala Thr Ser Asn Thr Asn Cys Arg 150 155 160 AAA CAA AGT CCC AGA AAT CGC CTA TGG TTG TTG ACC ATC CTT GTT TTG 583 Lys Gln Ser Pro Arg Asn Arg Leu Trp Leu Leu Thr Ile Leu Val Leu 165 170 175 TTA ATT CCA CTT GTA TTT ATA TAT CGA AAG TAC CGG AAA AGA AAG TGC 631 Leu Ile Pro Leu Val Phe Ile Tyr Arg Lys Tyr Arg Lys Arg Lys Cys 180 185 190 TGG AAA AGG AGA CAG GAT GAC CCT GAA TCT AGA ACC TCC AGT CGT GAA 679 Trp Lys Arg Arg Gln Asp Asp Pro Glu Ser Arg Thr Ser Ser Arg Glu 195 200 205 210 ACC ATA CCA ATG AAT GCC TCA AAT CTT AGC TTG AGT AAA TAC ATC CCG 727 Thr Ile Pro Met Asn Ala Ser Asn Leu Ser Leu Ser Lys Tyr Ile Pro 215 220 225 AGA ATT GCT GAA GAC ATG ACA ATC CAG GAA GCT AAA AAA TTT GCT CGA 775 Arg Ile Ala Glu Asp Met Thr Ile Gln Glu Ala Lys Lys Phe Ala Arg 230 235 240 GAA AAT AAC ATC AAG GAG GGC AAG ATA GAT GAG ATC ATG CAT GAC AGC 823 Glu Asn Asn Ile Lys Glu Gly Lys Ile Asp Glu Ile Met His Asp Ser 245 250 255

ATC CAA GAC ACA GCT GAG CAG AAA GTC CAG CTG CTC CTG TGC TGG TAC 871 Ile Gln Asp Thr Ala Glu Gln Lys Val Gln Leu Leu Leu Cys Trp Tyr 260 265 270 CAA TCT CAT GGG AAG AGT GAT GCA TAT CAA GAT TTA ATC AAG GGT CTC 919 Gln Ser His Gly Lys Ser Asp Ala Tyr Gln Asp Leu Ile Lys Gly Leu 275 280 285 290 AAA AAA GCC GAA TGT CGC-AGA ACC TTA GAT AAA TTT CAG GAC ATG GTC 967 Lys Lys Ala Glu Cys Arg Arg Thr Leu Asp Lys Phe Gln Asp Met Val 295 300 305 CAG AAG GAC CTT GGA AAA TCA ACC CCA GAC ACT GGA AAT GAA AAT GAA 1015 Gln Lys Asp Leu Gly Lys Ser Thr Pro Asp Thr Gly Asn Glu Asn Glu 310 315 320 GGA CAA TGT CTG GAG TGAAAACTAC CTCAGTTCCA GCCATGAAGA GAGGAGAGAG 1070 Gly Gln Cys Leu Glu 325 CCTGCCACCC ATGATGGAAA CAAAATGAAT GCCAACTGTA TTGACATTGG CAACTCCTGG 1130 TGTGTTCTCT TTGCCAGCAA ATGGTAGTTG ATACTCAGTG AGGGTCAAAT GACTAGCAGG 1190 TTCCAGGGAC TGCTTCTGTT ATTCTCTGCA GTTGCTGAGA TGAACCATTT TCTCTGTCTA 1250 CTGCAATTTT TACATTCAAA TGTCCATGAA ATTTGTATTA AATGTGAAGT GGAATCTGCA 1310 GTGTTTGTGT TTATATTCAT ATACTATGAA CTGAGGAGAA TTATAAACTG AAACAAATAC 1370 TCGCAGTTAA TTGAAGACCT TCCATTGATG GACAGTTCTT TTCCTCTCTA TATGGAAATG 1430 TATAATAGAA GAAATAATTT TTAAATTAAA GTATCTCTTT TTGCATTTCA AAAAAAAAAA 1490 AAAAAAAAAA AAAAAA 1506 (2) INFORMATION FOR SEQ ID NO : 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 327 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2: Met Leu Trp Ile Trp Ala Val Leu Pro Leu Val Leu Ala Gly Ser Gln 1 5 10 15 Leu Arg Val His Thr Gln Gly Ser Asn Ser Ile Ser Glu Ser Leu Lys 20 25 30 Leu Arg Arg Arg Val His Glu Thr Asp Lys Asn Cys Ser Glu Gly Leu 35 40 45 Tyr Gln Gly Gly Pro Phe Cys Cys Gln Pro Cys Gln Pro Gly Lys Lys 50 55 60 Lys Val Glu Asp Cys Lys Met Asn Gly Gly Thr Pro Thr Cys Ala Pro 65 70 75 80 Cys Thr Glu Gly Lys Glu Tyr Met Asp Lys Asn His Tyr Ala Asp Lys 85 90 95

Cys Arg Arg Cys Thr Leu Cys Asp Glu Glu His Gly Leu Glu Val Glu 100 105 110 Thr Asn Cys Thr Leu Thr Gln Asn Thr Lys Cys Lys Cys Lys Pro Asp 115 120 125 Phe Tyr Cys Asp Ser Pro Gly Cys Glu His Cys Val Arg Cys Ala Ser 130 135 140 Cys Glu His Gly Thr Leu Glu Pro Cys Thr Ala Thr Ser Asn Thr Asn 145 150 155 160 Cys Arg Lys Gln Ser Pro Arg Asn Arg Leu Trp Leu Leu Thr Ile Leu 165 170 175 Val Leu Leu Ile Pro Leu Val Phe Ile Tyr Arg Lys Tyr Arg Lys Arg 180 185 190 Lys Cys Trp Lys Arg Arg Gln Asp Asp Pro Glu Ser Arg Thr Ser Ser 195 200 205 Arg Glu Thr Ile Pro Met Asn Ala Ser Asn Leu Ser Leu Ser Lys Tyr 210 215 220 Ile Pro Arg Ile Ala Glu Asp Met Thr Ile Gln Glu Ala Lys Lys Phe 225 230 235 240 Ala Arg Glu Asn Asn Ile Lys Glu Gly Lys Ile Asp Glu Ile Met His 245 250 255 Asp Ser Ile Gln Asp Thr Ala Glu Gln Lys Val Gln Leu Leu Leu Cys 260 265 270 Trp Tyr Gin Ser His Gly Lys Ser Asp Ala Tyr Gln Asp Leu Ile Lys 275 280 285 Gly Leu Lys Lys Ala Glu Cys Arg Arg Thr Leu Asp Lys Phe Gln Asp 290 295 300 Met Val Gln Lys Asp Leu Gly Lys Ser Thr Pro Asp Thr Gly Asn Glu 305 310 315 320 Asn Glu Gly Gln Cys Leu Glu 325 (2) INFORMATION FOR SEQ ID NO : 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 40 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3: ACCCCTGTAC CCATTAAGTA CCCGACGACC GGGTACCGCG 40

(2) INFORMATION FOR SEQ ID NO : 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: unknown (D) TOPOLOGY: unknown (ii) MOLECULE TYPE :-DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4: GCGGGATCCT TAAAGCTTAT ACAAGCCG 28