CA2275438A1 - Improved methods for transducing cells - Google Patents

Abstract

Disclosed are compositions and methods for inhibiting the expression and/or activity of endogenous .beta.-interferon in cells targeted for transduction with viral vectors, particularly adenoviral vectors. Therefore, also provided are improved methods for treatment of genetically-based diseases by gene therapy. Also disclosed are methods for the treatment of neovascularization-related diseases, for examples, cancer, by the production in vivo of angiostatin, which inhibits the formation of new blood vessels. In particular embodiments, this is accomplished by transduction of macrophages ex vivo with a GM-CSF gene, thereby inducing the secretion of macrophage metalloelastase, which converts plasminogen to angiostatin. The transduced macrophages, when administered, naturally home to tumor sites to effectively localize the therapeutic effect.

Description

DESCRIPTION
IMPROVED METHODS FOR TRANSDUCING CELLS
BACKGROUND OF 'CHE INVENTION
The present application claims the priority of co-pending U.S. Provisional Patent Application Serial No. 601031,330, filed November 20, 1996, the entire disclosure of which is incorporated herein by reference without disclaimer. The government owns rights in the present invention pursuant to grant numbers R35-CA 42107 and CA-16672 from the National Institutes of Health.
1. Field of the Invention The present invention relates generally to the fields of cellular and molecular biology.
More particularly, it concerns compositions and methods for the transduction of cells, as well as compositions and methods for treating disease, including, but not limited to, cancer.

2. Description of Related Art Recent years have seen numerous advancements in the diagnosis and treatment of genetically-based disease. This advance has been due in large part to the revolution in molecular biology, which has allowed the genes responsible for a number of diseases to be cloned, sequenced, and manipulated in vitro. Using these techniques, the structure-function relationship of the proteins encoded by a number of these genes has been elucidated, allowing for the rational design of drugs which ameliorate the disease state.
A number of genes associated with particular diseases have one or more mutations which leads to the disease phenotype. This knowledge has led to the advent of gene therapy. In one aspect of gene therapy, a defective copy of a particular gene is replaced in vivo by the wild type copy of the desired gene. In other aspects of gene therapy, (i) cells can be engineered to express antigens which target the host cells for immune attack, (ii) a heterologous therapeutic gene can be introduced into cells, or (iii) the recombinant host cells can be "tagged"
with specific genetic markers to provide a method of tracking the fate of the "tagged" cells. Gene therapy protocols may also be designed to remedy diseases which are due to defects in multiple genes. Thus, gene therapy holds great promise for the future of curing a wide variety of diseases.
However, the high expectations for genetic therapy have yet to be fully realized (Marshall, 1995). A major obstacle to successful in vivo gene therapy is low transduction efficiency, or the efficiency of the insertion and expression of the selected transgene in host cells (Knowles et al., 1995; Grubb et al., 1994). Although transgene expression can be increased by administration of a high dose of vectors, the accompanying severe local inflammatory response may limit the effectiveness of increasing dosage. Also, the natural antiviral defense mechanisms of cells, which include production of interferons, can limit clinical effectiveness (Simon et al., 1993; Yei et al., 1994; Brody et al., 1994; Ginsberg et al., 1991 ). Therefore, the obstacles to successful gene therapy apparently are not fundamental in nature, but rather involve the need to properly deliver currently existing vectors {Marshall,1995). The ability to transduce cells in vivo with viral vectors containing one or more selected transgenes would represent an important advancement in the field of gene therapy.
Currently, cancer is one of the main targets in gene therapy trials. Once the diagnosis of cancer is established, the most urgent question is whether the disease is localized, or has spread to lymph nodes and distant organs. The most fearsome aspect of cancer is metastasis, and this fear is well justified. In nearly 50% of patients, surgical excision of primary neoplasms is not curative because metastasis has occurred by that time (Sugarbaker, 1977, 1979; Fidler and Balch, 1987).
Metastases can be located in different organs and in different regions of the same organ, making complete eradication by surgery, radiation, drugs, or biotherapy difficult.
Moreover, the organ microenvironment significantly influences the response of tumor cells to therapy (Fidler, 1995), as well as the efficiency of anticancer drugs, which must be delivered to tumor foci in amounts sufficient to destroy cells without leading to undesirable side effects (Fidler and Poste, 1985) Another major barrier to the treatment of metastases is the biological heterogeneity of cancer cells, and the rapid emergence of tumor cells with resistance to most conventional anticancer agents (Fidler and Poste, 1985). The design of more effective therapy for metastatic disease therefore requires a better insight into the molecular mechanisms that regulate the pathobiology of the process. One of the processes involved in the growth of both primary and secondary (metastatic) tumors is neovascularization, or creation of new blood vessels which grow into the tumor. This neovascularization is termed angiog;enesis (Folkman, 1986, 1989), which provides S the growing tumor with a blood supply and essential nutrients. Although tumors 1-2 mm in diameter can receive all nutrients by diffusion, further growth depends on the development of an adequate blood supply through angiogenesis. Inhit~ition of angiogenesis provides a novel and more general approach for treating metastases by manipulation of the host microenvironment.
The induction of angiogenesis is mediated by several angiogenic molecules released by both tumor cells and the normal cells surrounding tJne tumor cells. The prevascular stage of a tumor is associated with local benign tumors, whereas the vascular stage is associated with tumors capable of metastasizing. Moreover, studies using light microscopy and immunohistochemistry concluded that the number and density of microvessels in different human cancers directly correlate with their potential to invade and produce metastasis (Weidnc~r et al., 1991, 1993). Not all angiogenic tumors produce metastasis, but the inhibition of angiogenesis prevents the growth of tumor cells at both the primary and secondary sites and thus can prevent the emergence of metastases.
Often, the metastases are too small to be detected (<5 mm in diameter), and the primary neoplasm is surgically resected with curative intent. Unfortunately, the clinical reality is quite different. The resection of some primary neoplas~ms can lead to the accelerated growth of their distant metastases (Tyzzer, 1913; Sugarbaker ei' al., 1977; Gorelik et al., 1978, 1980, 1981;
Fisher et al., 1989). This accelerated growth is independent of specific immune response (Prehn, 1991, 1993) and has been termed concomitant honor resistance. Over the last 80 years, many different hypotheses for concomitant tumor resistance have emerged. These include the mechanical release of a large number of tumor cells during the surgical resection procedure, the sudden availability of "nutrients" for growth of metastases, the immunosuppressive effects of anesthesia and surgery that facilitate the escape of tumor cells from surveillance mechanisms, increased adhesive properties of platelets and blood coagulability, which aid the survival of circulating tumor emboli, and the production of a mitotic inhibitor by the local tumor (Sugarbaker et al., 1977; Gorelik,1983a,b; Prehn,1993).

WO 98/22605 PCT/~JS97/21475 However, recent studies have suggested a compelling explanation for this phenomenon.
O'Reilly et al. (1994) reported that in mice bearing Lewis lung carcinoma (3LL) subcutaneously (s.c.), the primary or local tumor releases an angiogenesis-inhibiting substance, named angiostatin. Angiostatin is a 38-kDa fragment of plasminogen that selectively inhibits proliferation of endothelial cells. Angiostatin suppresses vascularization and, hence, growth of lung metastases. Several studies have produced results consistent with this model. After systemic administration, purified angiostatin can produce apoptosis in metastases (Holmgren et al., 1995) and sustain dormancy of several human tumors implanted subcutaneously in nude mice (O'Reilly et al. , 1996). However, although it is known that angiostatin can be generated in vitro from plasminogen by digestion with pancreatic elastase (O'Reilly, 1994), how it is generated in vivo in tumors remains unclear. The ability to produce angiostatin in tumors in vivo would thus provide a significant advance in the art.
SUMMARY OF THE INVENTION
IS
The present invention seeks to overcome the limitations present in the prior art by providing compositions and methods for the efficient transduction of cells with nucleic acid constructs administered to host cells, for example using viral vectors. This is accomplished through a reduction of the endogenous expression and/or activity of /3-interferon in the cells which are targeted for transduction. Accordingly, the present invention provides improved methods for the treatment of genetically-based diseases by gene therapy, through the use of the improved transduction methods comprising reducing endogenous ~3-interferon expression and/or activity.
Also provided are methods for transduction of cells for the in vitro production of selected proteins or peptides.
The instant invention further provides methods for treating diseases associated with neovascularization, including genetically based diseases such as ADA
deficiency, cystic fibrosis, hemophilia and familial hypercholesterolemia, vascular proliferative diseases such as infantile hemangioma, arthritis, psoriasis and pulmonary hypertension, and various forms of cancer. This is achieved through the production in vivo of angiostatin, which inhibits the formation of new blood vessels.

The present invention provides compositions comprising a cell in which the endogenous expression and/or activity of (3-interferon is inhibited. The cells of the instant compositions may be cells in which the levels of endogenous ~3-interi:eron are high, such as macrophages.

The ~3-interferon inhibited cells provided in the present invention may further comprise an exogenous DNA segment. In certain aspects, the exogenous DNA segment may comprise an isolated gene encoding a selected protein or peptide. In other aspects, the DNA segment may comprise a ribozyme.
The exogenous DNA segments may be in operable relation to control sequences, such as promoters and enhancers, which direct the expression of a heterologous gene, such as GM-CSF
or elastase. In other embodiments of the present invention, the gene may be in operable relation and in reverse orientation to a promoter, whereby said promoter directs the production of an 1 S antisense transcript.
Thus the cells of the present invention are contemplated for use in the formulation of an anti-cancer therapeutic. The invention therefore provides for the use of the instant cells in the manufacture of a medicament for treating cancer.
The present invention also provides compositions for transducing a cell which can be characterized as including a ~i-interferon inhibitory factor which inhibits the activity of (3-interferon, and a heterologous DNA segment.
The (3-interferon inhibitory factors provided in the present invention may comprise an antibody which is immunologically reactive with ~i-interferon. Alternatively, the ~i-interferon inhibitory factors may comprise an isolated DNE1 sequence comprising a gene which encodes (3-interferon in operable relation and in reverse orientation to a promoter, whereby said promoter directs the production of an antisense transcript. In other aspects, the ~3-interferon inhibitory factor comprises an inhibitor of (3-interferon related protein kinase. In still other embodiments, the (3-interferon inhibitory factor comprises a ribo~zyme. In certain aspects of the invention, the (3-interferon inhibitory factor comprises a combination of two or more of an antibody which is immunologically reactive with (3-interferon, a gene which encodes (3-interferon in operable relation and in reverse orientation to a promoter, whereby said promoter directs the production of an antisense transcript, an inhibitor of (3-interferon related protein kinase and a ribozyme.
S The transducing compositions of the instant invention may include DNA
segments which comprise an isolated gene encoding a selected protein or peptide, for example GM-CSF or elastase. The DNA segments intended for use in expression will be in operable relation to a promoter that directs the expression of the selected protein or peptide. Thus expression vectors form another aspect of the present invention, particularly where the expression vector is a viral vector. In alternative embodiments, the expression vector may be an adenoviral or retroviral vector, preferably a replication defective adenoviral vector which is contained within a viral particle.
In other compositions, the DNA segment may be in operable relation and in reverse orientation to a promoter, whereby said promoter directs the expression of an antisense transcript.
In certain aspects of the present invention, the instant compositions are contemplated for use in rendering a cell susceptible to DNA uptake. In further embodiments, the present compositions are contemplated for use in sensitizing a cell to viral DNA
uptake. In still other embodiments, the present compositions are contemplated for use in transducing a cell with a DNA segment. Thus, in preferred aspects, the present invention provides for the use of the instant compositions in the preparation of a cell transduction/cell infection formulation. In additional aspects, the invention provides for the use of the instant compositions in the preparation of a transducing formulation for providing a DNA segment to a cell.
In further aspects of the invention, the compositions are contemplated for use in transducing a tumor cell with a tumor cell cytotoxic DNA segment. In certain preferred embodiments, the invention provides for the use of the instant compositions in the preparation of a medicament for treating cancer.

The present invention also provides improved methods of transducing a cell, comprising contacting the cell with a transducing composition that may be characterized as including a ~i-interferon inhibitory factor which inhibits the activity of (3-interferon, and a heterologous DNA
segment.
In the transducing methods provided herein, the (3-interferon inhibitory factor may be administered at a time effective for the transduction of said cell. The time of administration may be any reasonable time from before transduction to after transduction. In certain aspects of the present invention, the time of administration is from about 24 hours before the time of transduction to about 48 hours after transduction. In other embodiments, the time of administration may be variously from about 24 hours, about 18 hours, about 12 hours, about 6 hours or about 3 hours before the time of transduction to about 3 hours, about 6 hours, about 12 hours, about 20 hours, about 24 hours, about 30 hours, about 36 hours, about 40 hours, about 44 hours or about 48 hours after transduction. In other aspects, the time of administration may be from about 24 hours, about 18 hours, about 12 hours, about 6 hours or about 3 hours before the time of transduction up to and including the time of transduction.
Alternatively, the time of administration may be from the time of transduction to about 3 hours, about 6 hours, about 10 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours or about 48 hours after the time of transduction. It will be understood that the times are approximate, and therefore times which vary by up to one or two hours in either direction still fall within the spirit and scope of the invention.
In other methods of the present invention, the DNA segment may further comprise an isolated gene encoding a selected protein or peptide, such as GM-CSF, elastase, IRF-2, ADA, CFTR or ornithine transcarbamoylase. The DNA., segments intended for expression will be in operable relation to control sequences, such as promoters and enhancers.
In still other methods, the DNA segment may be in operable relation and in reverse orientation to a promoter, whereby said promoter directs the expression of an antisense transcript, such as (3-interferon antisense transcript.

WO 9$/22605 PCT/US97/21475 In certain methods provided in the instant disclosure, the DNA segment may be further defined as an expression vector, and in particular aspects a viral vector. In other methods, the vector may be an adenoviral vector, particularly a replication defective adenoviral vector. In certain embodiments, the adenoviral vector may be contained in an adenovirus.
In the transducing methods of the present invention, the target cell may be, for example, a macrophage, a tumor infiltrating lymphocyte, a tumor cell, an endothelial cell, a peripheral blood mononuclear cell or a stem cell. In further embodiments, the cell may be located within an animal, and in still further aspects, the animal may be a human subject.
In the improved transduction methods provided in the present invention, the ~3-interferon inhibitory factor may comprise an polyclonal or monoclonal antibody which is immunologically reactive with ~3-interferon. In other embodiments, the ~-interferon inhibitory factor may comprise an isolated DNA sequence comprising a gene, such as (3-interferon or IRF-1, which is in operable relation and in reverse orientation to a promoter, whereby said promoter directs the production of an antisense transcript. In still other methods, the (3-interferon inhibitory factor may comprise an inhibitor, such as a peptide or small chemical compound, of a J3-interferon-associated kinase. In further embodiments, the ~i-interferon inhibitory factor may comprise a ribozyme.
Thus the present invention provides improved methods for treating genetically-based diseases, which may comprise contacting a cell with a therapeutic transducing composition that may be characterized as including a (3-interferon inhibitory factor which inhibits the activity of (3-interferon, and a DNA segment comprising a therapeutic gene. Treatment is achieved by contacting the target cell with therapeutically effective amounts of the (3-interferon inhibitory factor which inhibits the activity of ~3-interferon, and a DNA segment comprising a therapeutic gene. Therapeutically effective amounts are those amounts which result in improved rates of transduction of the cell.
The present invention also provides a method of inhibiting a tumor in an animal comprising contacting the tumor with a therapeutically effective amount of a GM-CSF protein or peptide.

The present invention also provides methods for producing angiostatin in vivo, which may be characterized as including the steps of first contacting a macrophage with a transducing composition comprising a (3-interferon inhibitory factor which inhibits the expression or activity of (3-interferon, and a DNA segment comprising a GM-CSF gene, and second administering the transduced cell to an animal, wherein the tramsduced macrophage is stimulated by the transduced GM-CSF gene to secrete macrophage metalloc;lastase, thereby producing angiostatin from plasminogen.
The present invention also provides alternaitive methods for producing angiostatin in vivo, which may be characterized as including the steps of first contacting a cell other than a macrophage with a transducing composition comprising a ~3-interferon inhibitory factor which inhibits the expression or activity of (3-interferon., and a DNA segment comprising an elastase gene, preferably macrophage derived metalloelastase, and second administering the transduced cell to an animal, wherein the tramsduced cell secretes elastase, thereby producing angiostatin from plasminogen.
Thus, methods of treating diseases associated with neovascularization are provided which may comprise administering a cell which has been engineered to produce angiostatin in vivo to an animal in need of inhibition of neovascularization.
The present invention also provides methods of inhibiting a tumor in an animal which may comprise contacting the tumor with a therapeutically effective amount of a tumor inhibiting composition comprising a DNA segment comprising at least a first DNA sequence which encodes a GM-CSF or an elastase protein or peptide. In certain methods, the DNA segment may be further defined as an expression vector, which may further be contained in an adenovirus. In alternative methods, the vector is contained in a host cell. Thus the present invention provides tumor inhibiting compositions which may be viruses or transduced cells. The present invention thus provides for the use of a composition compriising a GM-CSF or elastase protein, peptide or nucleic acid in the manufacture of a medicament f ~r treating cancer.

WO 98!22605 PCT/US97/21475 The present invention provides methods for ex vivo cancer therapy, wherein a host cell is removed from an animal, contacted with a tumor inhibiting composition, and returned to the animal. In certain methods, the host cell may be a tumor infiltrating lymphocyte, a macrophage or a tumor cell. The present invention also provides methods for in vivo cancer therapy, wherein 5 the host cell is located within an animal, particularly a human subject. In certain aspects, the tumor inhibiting composition may further comprise a ~3-interferon inhibiting factor. In other embodiments of the present invention, the tumor inhibiting composition may be formulated for parenteral, intravenous, subcutaneous or oral administration.

10 The compositions that are intended for administration to an animal, such as the transducing compositions, the tumor inhibiting compositions and the viral and cell therapeutic compositions, may be dispersed in a pharmaceutically acceptable Garner.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. lA, FIG. 1B, FIG. 1C and FIG. 1D. Effect of anti-IFN-~i antibody on transgene expression. FIG. lA: Macrophages. FIG. 1B: NIH 3T3 cells. FIG.1C: Macrophages plus anti-IFN-j3. FIG. 1D: NIH 3T3 cells plus anti-IFN-~3. Cells were plated at the density of 105 cells/38-mm2 well. After 16 h (37°C), the cells were exposed for 1 h to increasing concentrations (PFU/cell) of AdSCMV-LacZ dispersed in 50 ml/well of serum-free Dulbecco's modified essential medium (DMEM)/F12 medium. DMEM with 10% fetal bovine serum (DMEM-10% FBS) (open circles) or DMEM-10% FBS containing 10 NU/ml of anti-IFN-~ antibody (filled squares) or 0.25 mg/ml rat IgG (open triangles) was then added to a final volume of 200 ml/well. The cultures were incubated at 37°C-5% C02 for an additional 48 h. (3-gal activity in the cells was then determined as described in Example 2. Dose-dependent enhancement of AdSCMV-LacZ infection of macrophages (FIG. 1 C) but not NIH 3T3 cells (FIG. 1 D) was seen using anti-IFN-(3. The cells were infected with 30 PFU AdSCMV-LacZ/cell as described above in the absence or presence of different concentrations of anti-IFN-(3 antibody (fil.led squares) or control rat IgG (open triangles).
*P<0.05 (Student's t test, two tailed).
FIG. 2. Fluorescence-activated cell sorting (FACS) analysis of ~3-gal activity.
Macrophages were incubated for 1 h with 30 PFU/cell AdSCMV-LacZ in 50 ml of serum-free DMEM/F 12 solution. One milliliter of DMEM/F 12-10% FBS or the medium containing 10 NU/ml of anti-IFN-(3 antibody or the medium containing 0.25 mg/ml of rat IgG
was then added.
The cells were cultured for 48 h under constant rocking. The ~i-gal activity was then determined.
FIG. 3A and FIG. 3B. Effect of exogenous IFN-~i on transgene expression in NIH

cells. FIG. 3A: NIH 3T3 cells were incubated for 1 h with 30 PFU AdSCMV-LacZ/cell.
Increasing concentrations of mIFN-(3 were then added to the cultures in medium alone (open circles) or in medium containing 10 NU/ml anti-IfN-(3 antibody (filled squares) or control rat IgG
(open triangles). (3-gal activity was measured 48 h later as described above with a reaction time of 10 min. FIG. 3B: Macrophages were plated at 5 x 104/ 38-mm2 well in a 96-well plate. Sixteen hours later, NIH 3T3 cells (5 x 104/ well) were seeded alone or onto the macrophage monolayers, and 2 to 4 h later, the cultures were infected with 3 x 106 PFU AdSCMV-LacZ/well in 50 ml of serum-free DMEM/F 12. One hour later, DMEM:/F 12-10% FBS (open squares) or the medium containing 10 NU/ml anti-IFN-/3 antibody (filled squares) was added to a final volume of 200 ml/well. The (3-gal activity was measured 48 h later as described above with the reaction time of 10 min for NIH 3T3 and NIH 3T3 + macrophages, and 60 min for macrophages cultured alone.
*P<0.05 (Student's t test, two tailed).
FIG. 4. Production of elastase by macrophages treated with LPS and IFN-y. PEM
were incubated in serum-free DMEM-F 12 alone or with LPS ( 100 ng/ml} and/or IFN-y ( 100 U/ml}.
The supernatants were collected at different times and assayed for elastinolytic activity as described in Example 4. The values are mean ~ S..D. of triplicate cultures.
- 30 FIG. SA and FIG. SB. Inhibition of elastase activity by LPS and IFN-y is independent of nitric oxide. FIG. SA: Nitrate content. FIG. SB: Elastase activity. PEM
were incubated in MEM containing 5% FBS (FIG. SA) or serum-free DMEM-F 12 (FIG. SB) alone or containing LPS ( 100 ng/ml), and/or IFN-y ( 100 U/ml) with or without 1 mM NMA. Culture supernatants were collected after 48 h and assayed for nitrite content (FIG. SA) or elastinolytic activity (FIG. SB) as described in Examples 5 and 4, respectively. The values are mean ~ S.D. of triplicate cultures.
FIG. 6A, FIG. 6B and FIG. 6C. Production of elastase by PEM treated with GM-CSF, M-CSF, or G-CSF. FIG. 6A: PEM treated with GM-CSF. FIG. 6B: PEM treated with M-CSF.
FIG. 6C: PEM treated with G-CSF. PEM were incubated in serum-free DMEM-F 12 alone or with different concentrations of recombinant GM-CSF (FIG. 6A), M-CSF (FIG.
6B), or G-CSF
(FIG. 6C). Culture supernatants were harvested after 48 h and assayed for elastinolytic activity.
The values are mean ~ S.D. of triplicate cultures. *p<0.05 compared to control macrophages.
**p<0.005 compared to control macrophages.
FIG. 7. GM-CSF enhances the stability of MME mRNA. PEM were incubated for 18 h in medium alone or medium containing GM-CSF (1000 U/ml). Cells were either collected at this time point (time 0), or actinomycin D was added in the medium to 10 ~g/ml and cells were collected at the indicated times (h) after the addition of the inhibitor. RNA
was extracted and separated on 1 % agarose, transferred onto a nylon membrane, and probed with 32P-labeled MME
and GAPDH cDNA probes. Data is calculated as the ratio of the area for the MME
transcript and the GAPDH transcript. Curves were fitted with the polynomial curve-fit program (order of 3) included in the Cricket graph computer software.
FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D. Macrophages expressed MME and generated angiostatin in 3LL-met s.c. tumor. FIG. 8A: Macrophage content. FIG. 8B: MME
mRNA
expression. FIG. 8C: MME activity. FIG. 8D: Angiostatin activity. Cells from subcutaneous 3 LL-met tumors was recultured and passaged successively in vitro. Macrophage content (FIG. 8A), MME mRNA expression (FIG. 8B), MME activity (FIG. 8C), and angiostatin activity (FIG. 8D) from each passage of the samples were analyzed.
FIG. 9A and FIG. 9B. 3LL tumor cells increased MME secretion and angiostatin production by macrophages. FIG. 9A: Purif ed mouse peritoneal macrophages, 3LL-nm, or 3LL-met cells were cultured in MEM-5% FBS separately or in combination for 24 hr. The cultures were rinsed briefly and incubated in serum-free DMEM/F 12 medium for 72 hr in the absence of human plasminogen. MME activity in the supernatants were determined. FIG. 9B:
Purified mouse peritoneal macrophages, 3LL-nm, or 3LL-met cells were cultured in MEM-5%
FBS separately or in combination for 24 hr. The; cultures were rinsed briefly and incubated in serum-free DMEM/F 12 medium for 72 hr in the presence of human plasminogen.
Angiostatin activity in the supernatants was determined.
FIG. l0A and FIG. lOB. Conditioned medium of 3LL tumor cells increased MME
secretion and angiostatin production. FIG. 10A: Purified mouse peritoneal macrophages were incubated for 24 hr in MEM containing 5% FBS or conditioned media of 3LL-nm or 3LL-met.
The cells were rinsed and cultured for 72 hr in serum-free DMEM/F 12 in the absence of human plasminogen. MME activity in the supernatants were then determined. FIG. lOB:
Purified mouse peritoneal macrophages were incubated for 24 hr in MEM containing 5% FBS
or conditioned media of 3LL-nm or 3LL-met. The cells were rinsed and cultured for 72 hr in serum-free DMEM/F 12 in the presence of human plasminogen. Angiostatin activity in the supernatants were then determined. Open box - medium cool; Hatched box -conditioned medium of 3LL-nm; Filled box - conditioned medium of 3LL-met.
FIG. 11A and FIG. 11B. Stimulation of MME secretion and angiostatin production by 3LL cell-conditioned medium was blocked by GM-CSF antibody. FIG. 11A: MME
activity.
FIG. 11B: Angiostatin activity. The conditioned medium of 3LL cells were incubated with 20 p,g/ml GM-CSF antibody or the same concentration of rat IgG for 45 min at 37°C and then used to treat macrophages as described in the legend t~~ FIG. l0A and FIG. IOB. MME
(FIG. 1 lA) and angiostatin (FIG. 11 B) activities in the supernatants were then determined.
FIG. 12A and FIG. 12B. Induction of Macrophage-mediated Angiostatin Production by B 16-F 10 Cells Engineered to Constitutively Relf~ase GM-CSF. FIG. 12A.
Elastase activity.
FIG. 12B. Inhibition of BCE. M~~ = macrophages; P = wild-type B 16-F10;
H = B 16-F 10-GMCSF(high); M = B I 6-F 10-GMCSF(medium); C = B-I 6-F 10-GMCSF(control).
FIG. 13A and FIG. 13B. Induction of Macrophage-mediated Angiostatin Production by K1735M2 cells Engineered to constitutively Release GMCSF. FIG. 13A.
Elastase activity.

FIG. 13B. Inhibition of BCE. P = wild type K1735M2, N = K1735M2-Neo, C = K1735M2-GMCSF(control); H = K1735M2-GMCSF(high); Mgr = macrophages.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
The present invention provides compositions and methods for the efficient transduction of cells with heterologous genes or cDNAs administered using viral vectors. This is accomplished through a reduction of the endogenous expression andlor activity of ~3-interferon in the target cells.
Thus, the present invention provides improved methods for the treatment of genetically-based diseases by gene therapy, through the use of the improved transduction methods disclosed herein.
The instant invention further provides methods for treating diseases associated with neovascularization, including, but not limited to, cancer. This is achieved through the production in vivo of angiostatin. In a particular embodiment of the present invention, macrophages are 1 S transduced ex vivo with GM-CSF, thereby activating the macrophages to produce macrophage metalloelastase (MME). The administered macrophages home to primary and metastatic tumor sites, where the MME produced by the macrophages converts plasminogen to angiostatin.
Gene therapy is an exciting development that has vast potential to treat a number of genetically-based diseases. The potential benefits of gene therapy have led to an increasing number of studies where a therapeutic gene is used to treat a variety of diseases. However, the high expectations for gene therapy have yet to be fully realized (Marshall,1995).
The initial reports on gene therapy as a therapeutic approach for treating disease used retroviral vectors to mediate gene transfer (Anderson, 1984). This system has been used to deliver a copy of the correct adenosine deaminase (ADA) gene to humans with a defective version of the gene. However, the rates of transfer and expression of genes using retroviral vectors can vary dramatically from patient to patient (Marshall, 1995).
Another system which has been used widely is adenovirus. However, while current methods using adenoviral vectors allow for the transduction of a number of different cell types in vitro, the results in vivo have been limited. The major obstacle to successful in vivo gene therapy is the low efficiency of the insertion and expression of the selected transgene in host cells.
Adenoviral vectors were used to treat mice with the rare recessive genetic disorder ornithine 5 transcarbamoylase (OTC) deficiency. While tile adenoviral constructs were successful in delivering the normal OTC gene in vivo, normal e~;pression of OTC was observed in only 4 of 17 instances (Stratford-Perricaudetet al., 1991 ). Therefore the defect was only partially corrected in most of the mice, and led to no phenotypic or physiological change.
10 Similarly, attempts have been made to treat cystic fibrosis in rats, mice and humans by using adenovirus to transfer the gene encodin;~ the correct version of the cystic fibrosis transmembrane regulator (CFTR) in vivo into the. affected columnar epithelial cells lining the airways of the lung (Rosenfeld et al., 1992; Grubb .et al., 1994; Knowles et al., 1995). While gene transfer using this technique was detected, in all cases the efficiency of transfer and expression was 15 too low to be of physiological relevance.
Although transgene expression can be increased by administration of a high dose of vectors, the accompanying severe local inflammatory response limits the effectiveness of increasing dosage.
Also, the natural antiviral defense mechanism of cells, which includes production of interferons, limits clinical effectiveness (Simon et al., 1993; 'i~ei et al., 1994; Brody et al., 1994; Ginsberg et al. , 1991 ). Therefore, the obstacles to successful gene therapy are apparently not fundamental in nature, but rather involve the need to properly delivE,r currently existing vectors (Marshall,1995).
Efforts to decipher the mechanism of diminshed transduction efficiency have focused on the interferons, which are a family of multifunctional proteins with potent antiviral activities (Sen and Ransohoff,1993; Sen and Lengyel, 1992; Gutte;rman, 1994). Among the 3 types of interferons (IFNs), a-interferon (IFN-a) and y-interferon (IFN-y} are mainly produced by leukocytes, whereas (3-interferon (IFN-(3) is produced by many cell types, including epithelial cells, fibroblasts and macrophages (Sen and Lengyel, 1992). Macrophages that express endogenous IFN-a and -~i are resistant to viral infection; however, this property can be compromised by antibodies against IFN-~3 (Belardelli et al., 1987b).

In addition, treatment of cells with exogenous IFN-(3 or IFN-inducing agents can suppress replication of DNA and RNA viruses (Sen and Ransohoff,1993). Constitutive expression of IFN-~3 can protect monocytes/macrophages against viral infections (Belardelli et al., 1987a;
Lallemand et al., 1996). Many tumors are infiltrated by macrophages that can produce S cytokines, including IFN-~3, subsequent to interaction with microorganisms or their products (Nathan, 1987). Whether the production of IFN-(3 by macrophages could protect bystander tumor cells from viral infection remains unclear.
In viewing the prior art, a significant question that remains unanswered is whether the reduction in endogenous ~i-interferon will allow the host cells to be transduced and to express significant levels of an introduced gene. Since the production of (3-interferon in response to viral infection is a natural response of a functional host cell, it is unclear if this process can be inhibited without having an adverse effect on other functions of the host cell, including protein production. It is entirely possible that blocking of ~3-interferon will result in rapid cell death, apoptosis or shut off of host cell synthetic machinery.
Another limitation present in the prior art is the lack of data concerning the duration of the (3-interferon inhibition necessary to protect cells against viral infection. Since protection against undesired viral infection would be compromised when ~i-interferon is inhibited indefinitely, the prolonged inhibition of ø-interferon is of limited clinical significance.
The present invention discloses that the transduction efficiency of AdSCMV-LacZ is inversely correlated with expression of endogenous IFN-Vii. Moreover, the presence of anti-IFN-(3 antibody during the infection phase can increase transduction efficiency and block the inhibition of exogenous IFN-~i on cells which have low IFN-(3 expression. As used herein, transduction does not include viral replication, but does include expression of non-viral genes by the vector.
Further, the inventors' studies suggest that in vivo transduction of tumors may be inhibited by IFN-(3 produced by infiltrating macrophages. This "protection of bystander cells"
from infecting viruses has particular relevance for gene therapy.

Thus, a particular embodiment of the present invention concerns methods and compositions for the efficient transduction of cells which express (3-interferon, or which are located in proximity to cells which express (3-interferon. This is accomplished through the reduction of (3-interferon expression, thus rendering cells amenable to transduction.
Further particular embodiments of the present invention concern methods and compositions for the efficient transduction of cells in vitro which express (3-interferon, or which are located in proximity to cells which express ~i..interferon. The cells thus transduced can then be used to produce heterologous proteins in vitro, or as compositions which are administered to an animal to produce a selected protein or peptide in vivo.
Additionally, certain aspects of the present invention concern methods and compositions for the efficient transduction of cells in vivo which express (3-interferon, or which are located in proximity to cells which express (3-interferon. Other aspects of the present invention concern the I S reduction of ~i-interferon expression in vivo for defined periods of time, thereby providing an improved procedure for transducing cells in vivo.
Further aspects of the present invention concern methods and compositions for treating a variety of diseases, including genetically based diseases such as ADA
deficiency, cystic fibrosis, hemophilia and familial hypercholesterolemia, v;~scular proliferative diseases such as infantile hemangioma, arthritis, psoriasis and pulmonary hypertension. A particular aspect of the present invention concerns methods and compositions for the treatment of cancer.
Once a diagnosis of cancer is established., the urgent question is whether the disease is localized to the primary site or whether it already spread to the regional lymph nodes and distant organs. The spread of cancer from the primary site to secondary locations, termed metastasis, is responsible for the majority of cancer related deaths. Cancer metastasis consists of multinte complex, interacting, and interdependent steps, each of which is rate-limiting, since a failure to complete any of the steps prevents the tumor cell from producing a metastasis.
The tumor cells that eventually give rise to metastases must survive a series of potentially lethal interactions with host homeostatic mechanisms. The balance of these interactions can vary among different patients with different neoplasms or even among different patients with the same type of neoplasm (Fidler, 1990, 1995).
The essential steps in the formation of a metastasis are similar in all tumors (Poste and Fidler, 1980; Fidler, 1990) and consist of the following. First, after neoplastic transformation, progressive proliferation of neoplastic cells is initially supported with nutrients supplied from the organ microenvironment by diffusion, and second, neovascularization or angiogenesis must take place for a tumor mass to exceed 1-2 mm in diameter. The synthesis and secretion of different angiogenic molecules and suppression of inhibitory molecules are responsible for the establishment of a capillary network from the surrounding host tissue. Third, some tumor cells can downregulate expression of cohesive molecules and have increased motility and, thus, can detach from the primary lesion. Invasion of the host stroma by some tumor cells occurs by several parallel mechanisms. Capillaries and thin-walled venules, like lymphatic channels, offer very little resistance to penetration by tumor cells and provide the most common pathways for tumor cell entry into the circulation. Fourth, detachment and embolization of single tumor cells or cell aggregates occurs next, the vast majority of circulating tumor cells being rapidly destroyed. Fifth, once the tumor cells have survived the circulation, they must arrest in the capillary beds of distant organs by adhering either to capillary endothelial cells or to exposed subendothelial basement membranes. Sixth, tumor cells (especially those in aggregates) can proliferate within the lumen of the blood vessel, but the majority extravasate into the organ parenchyma by mechanisms similar to those operative during invasion. Seventh, tumor cells bearing appropriate cell surface receptors can respond to paracrine growth factors and hence proliferate in the organ parenchyma. Eighth, the metastatic cells must evade destruction by host defenses that include specific and nonspecific immune responses. Ninth, to exceed a mass of 1-2 mm in diameter, metastases must develop a vascular network. The metastases can then give rise to additional metastases, i. e. , the phenomenon of "metastasis of metastases".
The term "cancer" embraces a collection of malignancies, with each cancer of each organ consisting of numerous subsets. Indeed, by the time of initial diagnosis, cancers consist of multiple subpopulations of cells with diverse genetic, biochemical, immunological and biological characteristics (Hart and Fidler, 1981 ). Biological heterogeneity is also prominent within and among metastases (Fidler, 1990). This heterogeneity, and the rapid emergence of tumor cells with resistance to conventional anticancer agents, is a major barrier to the treatment of metastases.
The design of a more effective therapy for metastatic disease therefore requires a better insight into the molecular mechanisms that regulate the pathobiology of the process. One of the obligate steps described above which is involved in the growth of both primary and secondary (metastatic) tumors is neovascularization, or creation of new blood vessels which grow into the tumor. This neovascularization is termed angio~;enesis (Folkman, 1986, 1989), which provides the growing tumor with a blood supply and essential nutrients.
Angiogenesis is mediated by multiple molecules that are released by both host cells and tumor cells. The host cells involved include endothelial cells, epithelial cells, mesothelial cells and leukocytes. Angiogenesis consists of sequential processes emanating from microvascular endothelial cells (Folkman, 1986). To generate capillary sprouts, endothelial cells must proliferate, migrate and penetrate host stroma, with the direction of migration generally pointing toward the source of angiogenic molecules.
There is a growing body of evidence 'that tumor growth is angiogenesis-dependent (O'Reilly et al., 1996). A number of angiogenesis inhibitors have been studied as possible anticancer agents. Recently, a substance termed angiostatin has been described which suppresses vascularization, and hence, growth of both primary and secondary (metastatic) tumors (O'Reilly et al., 1994). Studies have shown that after systemic administration, purified angiostatin can produce apoptosis in metastases (Holmgren et al. , 1995) and sustain dormancy of several human tumors implanted subcutaneously in nude mice (CfReilly et al., 1996).
Angiostatin is a 38 kDa fragment of plasminogen, and can be generated from plasminogen in vitro by digestion with pancreatic elastase. However, how angiostatin is produced in vivo from circulating plasminogen has remained unanswered. As described above, purified angiostatin has been used to treat tumors in vivo, however, due to the short circulating _ 30 half life of angiostatin, the treatments must be given twice daily for the duration of the regimen.
An additional limitation of the angiostatin treatments of the prior art is that the administration of angiostatin is systemic. Since a number of normal and critical cellular functions (including, but not limited to, wound healing) are dependent on angiogenesis, systemic, long-term inhibition of angiogenesis could be deleterious to the host organism.
Therefore, provided in the present invention are methods and compositions for the in vivo 5 production of angiostatin which remove the proposed requirement of twice daily administration.
Additionally, in certain embodiments, the production of angiostatin is concentrated in the tumors targeted for treatment, thus limiting the systemic effects of the inhibition of angiogenesis.
In certain aspects of the present invention, angiostatin is produced by elastase, preferably 10 macrophage metalloelastase (MME), which is induced in macrophages by granulocyte-macrophage colony stimulating factor (GM-CSF). The inventors' studies of two tumor systems in an art-accepted animal model, show that primary subcutaneous tumor cells transduced with therapeutically effective amounts of GM-CSF, recruit macrophages into the tumor lesion and stimulate MME expression in the infiltrating macrophages. The MME in turn degrades 15 plasminogen to angiostatin that circulates into distant capillary beds where it suppresses angiogenesis and hence suppresses the growth of distant metastases.
I. Inhibition of (3-interferon 20 As described herein, numerous aspects of the present invention concern the inhibition of (3-interferon expression and/or the neutralization of the action of ~3-interferon, either in cells targeted for transduction or in bystander cells proximal to cells targeted for transduction. The inhibition can be in vitro or in vivo, depending on the particular aspect of the invention practiced.
Further, the inhibition of (3-interferon expression can be transient or sustained for discrete periods of time.
There are a variety of methods for accomplishing the inhibition of (3-interferon expression, including, but not limited to, the use of anti-~i-interferon antibodies, antisense methodology, particularly with regards to ~i-interferon itself and interferon regulatory factor-1, ribozymes, inhibition of enzymes involved in the (3-interferon transduction pathway, including the protein kinases associated with the ~3-interferon receptor binding proteins, overexpression of interferon regulatory factor-2 and homologous recombination. Each of these methods are discussed in greater detail below.
A. Anti-(3-IFN antibodies As discussed above, antibodies to (3-interferon can make cells less resistant to viral infection. In certain embodiments of the present invention, anti-(3-interferon antibodies are administered with the transducing construct to effect efficient transduction of the target cells.
A number of different anti-~3-interferor.~ antibodies have been described and are commercially available. For example, a purified rabbit polyclonal anti-(3-interferon antibody can be purchased from Lee BioMolecular Research, Inc. (California), and a rat monoclonal anti-~3-interferon antibody can be purchased from Yamasa~. Shoyu Co., Inc. (Japan).
Additionally, polyclonal or monoclonal antibodies can be produced using techniques well known to those of skill in the art, as described below.
1. Antibody Production In certain aspects of the present invention., the preparation of polyclonal or monoclonal antibodies, for example to ~i-interferon, is contemplated. Means for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988; incorporated herein by re;ference).
The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclanal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition in accordance with the present invention, either with or without prior i.mmunotolerizing, depending on the antigen composition and protocol being employed, and colJlecting antisera from that immunized animal.
A wide range of animal species can be used for the production of antisera.
Typically the animal used for production of anti-antisera is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
As is also well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Suitable adjuvants include all acceptable immunostimulatory compounds, such as cytokines, toxins or synthetic compositions.
IS
Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12, g-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (C WS) in a 2% squalene/Tween 80 emulsion. MHC antigens may even be used.
Exemplary, often preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
In addition to adjuvants, it may be desirable to coadminister biologic response modifiers (BRM), which have been shown to upregulate T cell immunity or downregulate suppressor cell activity. Such BRMs include, but are not limited to, Cimetidine (CIM; 1200 mgld) (Smith/Kline, PA); or low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ) and Cytokines such as g-interferon, IL-2, or IL-12 or genes encoding proteins involved in immune helper functions, such as B-7.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immuno;;en (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization.
A second, booster injection, may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serurr~ isolated and stored, and/or the animal can be used to generate MAbs.
For production of rabbit polyclonal antibodies, the animal can be bled through an ear vein or alternatively by cardiac puncture. The removed blood is allowed to coagulate and then centrifuged to separate serum components from vcrhole cells and blood clots.
The serum may be used as is for various applications or else the desired antibody fraction may be purified by well-known methods, such as affinity chromatography using another antibody, a peptide bound to a solid matrix, or by using, e.g., protein A or protein G chromatography.
MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, incorporated herein by reference.
Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified (3-interferon protein, polypeptide or peptide, or any ~i-interferon composition, if used after tolerization to common antigens. The immunizing composition is administered in a manner effective to stimulate antibody producing cells.
The methods for generating monoclonal antibodies (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, pp. 60-61 ), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

The animals are injected with antigen, generally as described above. The antigen may be coupled to carrier molecules such as keyhole limpet hemocyanin if necessary.
The antigen would typically be mixed with adjuvant, such as Freund's complete or incomplete adjuvant.
Booster injections with the same antigen would occur at approximately two-week intervals.
Following immunization, somatic cells with the potential for producing antibodies, specifically B lymphocytes (B cells), are selected for use in the MAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately S x 107 to 2 x 108 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, pp. 65-66, 1986; Campbell, pp. 75-83, 1984). cites). For example, where the immunized animal is a mouse, one may use P3-X63/AgB, X63-Ag8.653, NS1/l.Ag 4 1, Sp210-Ag 14, FO, NSO/LJ, MPC-11, MPC 11-X45-GTG 1.7 and S 194/SXXO Bul; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection with human cell fusions.

One preferred marine myeloma cell is the lVS-1 myeloma cell line (also termed Ag4-1 ), which is readily available from the NIGMS Human Genetic Mutant Cell Repository by requesting cell line repository number GM3573. Another mouse myeloma cell line that may be used is the 8-azaguanine-resistant mouse marine lnyeloma SP2/0 non-producer cell line.
S
Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a 2:1 proportion, though the proportion may vary from about 20:1 to about 1:1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes. Fusion 10 methods using Sendai virus have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al. ( 1977). The use of electrically induced fusion methods is also appropriate (Goding pp. 71-74, 1986).
Fusion procedures usually produce viable hybrids at low frequencies, about 1 x 10'6 to 15 1 x 10-8. However, this does not pose a problem. as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indef nitely) by culturing in a selective medium. The selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media. Exemplary and preferred agents are aminopterin, methotrexate, and azaserine.
20 Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT
medium).
Where azaserine is used, the media is supplemented with hypoxanthine.

25 The preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT mef~.ium. The myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxantlune phosphoribosyl transferase (HPRT), and they cannot survive. The B cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide MAbs. The cell lines may be exploited for MAb production in two basic ways.
A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion (e.g., a syngeneic mouse). Optionally, the animals are primed with a hydrocarbon, especially oils such as pristane (tetramethylpentadecane) prior to injection. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide MAbs in high concentration.
The individual cell lines could also be cultured in vitro, where the MAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
MAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
Fragments of the monoclonal antibodies of the invention can be obtained from the monoclonal antibodies so produced by methods which include digestion with enzymes, such as pepsin or papain, andlor by cleavage of disulfide bonds by chemical reduction.
Alternatively, monoclonal antibody fragments encompassed by the present invention can be synthesized using an automated peptide synthesizer.
It is also contemplated that a molecular cloning approach may be used to generate monoclonal antibodies. For this, combinatorial immunoglobulin phagemid libraries are prepared from RNA isolated from the spleen of the immunized animal, and phagemids expressing appropriate antibodies are selected by panning using cells expressing the antigen and control cells e.g., normal-versus-tumor cells. The advantages of this approach over conventional hybridoma techniques are that approximately 104 times as many antibodies can be produced and screened in a single round, and that new spc:cificities are generated by H and L chain combination which further increases the chance of finding appropriate antibodies.
For example, the gene encoding ~3-interferon (see below) can be subcloned into a baculoviral expression vector. The engineered baculoviral vector can then be used to infect insect cells, and then ~3-interferon can be purified from the insect cells an appropriate time after the induction of expression of the baculoviral vector. The purified (3-interferon can then be used to immunize animals with an appropriate adjuvant, following a standard immunization protocol.
After approximately 4-12 weeks, the anti-(3-interfc;ron antibodies can be purified from the sera of immunized animals.
B. Antisense In certain other embodiments of the present invention, antisense methodology is used to inhibit ~-interferon expression. In antisense constructs contemplated for use in the present invention, the gene encoding either (3-interferon or IRF-1 is subcloned into an appropriate expression vector, in operable relation to regulatory sequences as described in Section V below.
In certain aspects of the invention, these antisensf: constructs are administered to the target cells (Section III below) prior to administration of the viral vectors comprising heterologous genes (Section II below). In other aspects of the instant invention, the antisense constructs are comprised within viral vectors themselves.
Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (~3:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation;
targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide, for example (3-interferon, and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNAs, may be employed to inhibit (3-interferon gene transcription or translation or both within the cells of the present invention.
Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a (3-interferon gene. It is contemplated that effective antisense constructs will often include regions complementary to intron/exon splice junctions. Thus, antisense constructs with complementarity to regions within 50-200 bases of an intron-exon splice junction of (3-interferon are contemplated for use herewith. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether the expression of ~i-interferon and/or other genes having complementary sequences is affected.
"Antisense" or "complementary" means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme) could be designed.
These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.

It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. 7.'he cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
Particularly contemplated for use in the present invention are amtisense constructs directed to (3-interferon and interferon regulatory factor-1 (IRF-1 ).
1. ~i-interferon In particular aspects of the present invention, the antisense constructs are directed to (3-interferon itself. The cloning and sequencing of the human (Taniguchi et al., 1980; GenBank accession numbers J00218, K00616 and M1102'9) and mouse (Higashi et al., 1983;
GenBank accession numbers X 14455 and X 14029) (3-interferon genes have been described.
2. IRF-1 In other aspects of the present invention, p.-interferon expression is inhibited by antisense constructs to interferon regulatory factor-1. Interferon regulatory factor-1 (IRF-1) is a positive control factor which binds efficiently to repeated hexamer motifs present in the regulatory region of interferon genes (Miyamoto et al., 1988). Theae motifs operate as virus-inducible enhancers (Fujita et al., 1987), and indeed transcription of the IRF-1 gene itself is induced by viral infection. This suggests that induction of IFN genes upon viral infection may be due to increased transcription of the IRF-1 gene. Expression of an IRF-1 antisense RNA in the human fibroblast cell line GM-637 results in strong inhibition of IFN-(3 gene expression (Reis et al., 1992), although this was not found in HeLa cells (Pine et al., 1990). This suggests alternate pathways for the induction of the (3-interferon gene.
The IRF-1 gene has been cloned from human (GenBank accession number X 14454) and mouse (GenBank accession numbers M21065, M2 5560 and J03160).

C. Ribozymes Another method for inhibiting (3-interferon expression contemplated in the present invention is via ribozymes. Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor.
5 Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion.
Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cech, 1987; Gerlach et al., 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cech et al., 1981;
10 Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
Ribozyme catalysis has primarily been observed as part of sequence-specific 15 cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cech et al., 1981 ). For example, U.S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA
restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al., 1991;
Sarver et al., 1990;
20 Sioud et al. , 1992). Recently, it was reported that ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.
25 Several different ribozyme motifs have been described with RNA cleavage activity (Symons, 1992). Examples that are expected to function equivalently for the down regulation of (3-interferon include sequences from the Group I self splicing introns including Tobacco Ringspot Virus (Prody et al., 1986), Avocado Sunblotch Viroid (Palukaitis et al., 1979 and Symons, 1981 ), and Lucerne Transient Streak Virus (Forster and Symons, 1987).
Sequences 30 from these and related viruses are referred to as hammerhead ribozyme based on a predicted folded secondary structure.

Other suitable ribozymes include sequencfa from RNase P with RNA cleavage activity (Yuan et al. , 1992, Yuan and Altman, 1994), haipin ribozyme structures (Berzal-Herranz et al. , 1992 and Chowrira et al., 1993) and Hepatitis Delta virus based ribozymes. The general design and optimization of ribozyme directed RNA cleavage activity has been discussed in detail (Haseloff and Gerlach, 1988, Symons, 1992, Chov~~rira et al., 1994, and Thompson et al., 1995).
The other variable on ribozyme design is the selection of a cleavage site on a given target RNA. Ribozymes are targeted to a given sequence by virtue of annealing to a site by complimentary base pair interactions. Two stretches of homology are required for this targeting.
These stretches of homologous sequences flank the catalytic ribozyme structure defined above.
Each stretch of homologous sequence can vary in length from 7 to 1 S
nucleotides. The only requirement for defining the homologous sequences is that, on the target RNA, they are separated by a specific sequence which is the cleavage site. For hammerhead ribozyme, the cleavage site is a dinucleotide sequence on the target RNA is a uracil (U) followed by either an adenine, cytosine or uracil (A,C or U) (Perriman et al., 1992 and Thompson et al., 1995). The frequency of this dinucleotide occurring in any given RNA is statistically 3 out of 16.
Therefore, for a given target messenger RNA of 1000 bases, 18'7 dinucleotide cleavage sites are statistically possible.
The large number of possible cleavage sites in (3-interferon coupled with the growing number of sequences with demonstrated catalytic RNA cleavage activity indicates that a large number of ribozymes that have the potential to downregulate (3-interferon are available.
Designing and testing ribozymes for efficient cleavage of a target RNA is a process well known to those skilled in the art. Examples of scientific methods for designing and testing ribozymes are described by Chowrira et al. , ( 1994) and Lieber and Strauss ( 1995), each incorporated by reference. The identification of operative and preferred sequences for use in (3-interferon-targeted ribozymes is simply a matter of prepaJring and testing a given sequence, and is a routinely practiced "screening" method known to those of skill in the art.
D. Kinase Inhibitors ~i-interferon stimulates transcriptional activation in a variety of cells.
This activation is stimulated by the binding of ~3-interferon to cognate receptors on the surface of certain cell types.

Most of the elements of the interferon transduction pathway have been identified and sequentially ordered. The interferon receptor binding proteins do not contain a functional tyrosine kinase, but instead recruit Tyk2 and Jakl. These cytoplasmic tyrosine kinases are implicated in the phosphorylation of relevant cytoplasmic proteins, termed signal transducers and activators of transcription (STATs). These phosphorylated proteins then migrate to the nucleus to constitute a transcription factor which interacts with elements in the promoter of (3-interferon inducible genes.
In certain aspects of the present invention, inhibition of the tyrosine kinases associated with ~3-interferon transduction is accomplished by using small chemical compounds, peptides or peptidometic compounds which inhibit tyrosine kinase activity. Alternatively, any of the methods used herein to reduce gene expression, exemplified by, but not limited to, antisense, ribozymes and homologous recombination, are contemplated for use in the present invention to inhibit tyrosine kinase activity. In particular aspects of the present invention, the inhibition of I S the specific tyrosine kinases associated with ~i-interferon transduction is localized to a tumor being targeted for transduction.
The peptide compounds contemplated for use in the present invention may be synthesized using known methods for peptide synthesis (Atherton & Shepard, 1989). The preferred method for synthesis is standard solid phase methodology, such as that based on the 9-fluorenylmethyloxycarbonyl FMOC protecting group (Barlos et al., 1989), with glycine -functionalized o-chlorotrityl polystyrene resin. These methods are also adaptable to placement of linking units on the end of the compound to provide additional functionalities, as desired.
A particular advantage to the solid phase method of synthesis is the opportunity for modification of these compounds using combinatorial synthesis techniques.
Combinatorial synthesis techniques are defined as those techniques producing large collections or libraries of compounds by sequentially linking different building blocks. Libraries can be constructed using compounds free in solution, but preferably the compound is linked to a solid support such as a bead, solid particle or even displayed on the surface of a microorganism.

Several methods exist for combinatorial synthesis (Holmes et al., 1995;
Burbaum et al., 1995; Martin et al., 1995; Freier et al., 1995; Pei et al., 1991; Bruce et al., 1995; Ohlmeyer et al., 1993); however, the preferred methods are split synthesis or parallel synthesis. Split synthesis may be used to produce small amounts of a relatively large number of compounds, while parallel synthesis will produce larger amounts of a relatively small number of compounds. In general terms, using split synthesis, compounds are synthesized on the surface of a microparticle. At each step, the particles are partitioned into several groups for the addition of the next component.
The different groups are then recombined and partitioned to form new groups.
The process is repeated until the compound is completed. Each particle holds several copies of the same compound allowing for facile separation and purification. Split synthesis can only be conducted using a solid support.
An alternative technique known as parallel synthesis may be conducted either in solid phase or solution. Using parallel synthesis, diflEerent compounds are synthesized in separate 1 S receptacles, often using automation. Parallel synthesis may be conducted in microtiter plate where different reagents can be added to each well in a predefined manner to produce a combinatorial library. It is well understood that many modifications of this technique exist and can be adapted for use with the present invention.
E. Overexpression of IRF-2 Interferon regulatory factor-2 (IRF-2) is a. repressor of [3-interferon expression (Harada et al., 1989). IRF-2, an antagonistic repressor of IRF-1, represses the effect of IRF-1. IRF-2 apparently acts by competing for the same cis-acting recognition sequences as IRF-1. Whereas the expression of IRF-1 induces (3-interferon, the concomitant expression of IRF-2 represses this activity (Harada et al., 1989, 1990).
The IRF-2 gene has been cloned from human (GenBank accession number X 15949) and mouse (GenBank accession number J03 i 68). In pertain aspects of the present invention, IRF-2 is overexpressed in cells which are then transduced with a viral vector containing a selected gene.
The IRF-2 gene can be subcloned into an expression vector, many examples of which are known to those of skill in the art, and subsequently introduced to the cells targeted for transduction by conventional DNA transfer means described herein.

F. Homologous Recombination Another approach for inhibiting (3-interferon involves the use of homologous recombination, or "knock-out technology". Homologous recombination relies, like antisense, on S the tendency of nucleic acids to base pair with complementary sequences. In this instance, the base pairing serves to facilitate the interaction of two separate nucleic acid molecules so that strand breakage and repair can take place. In other words, the "homologous"
aspect of the method relies on sequence homology to bring two complementary sequences into close proximity, while the "recombination" aspect provides for one complementary sequence to replace the other by virtue of the breaking of certain bonds and the formation of others.
Put into practice, homologous recombination is used as follows. First, the target gene is selected within the host cell, in this case, (3-interferon. Sequences homologous to the ~3-interferon target gene are then included in a genetic construct, along with some mutation that will render the target gene inactive (stop codon, interruption, and the like). The homologous sequences flanking the inactivating mutation are said to "flank" the mutation.
Flanking, in this context, simply means that target homologous sequences are located both upstream (5') and downstream (3') of the mutation. These sequences should correspond to some sequences upstream and downstream of the target gene. The construct is then introduced into the cell, thus permitting recombination between the cellular sequences and the construct.
As a practical matter, the genetic construct will normally act as far more than a vehicle to interrupt the gene. For example, it is important to be able to select for recombinants and, therefore, it is common to include within the construct a selectable marker gene. This gene permits selection of cells that have integrated the construct into their genomic DNA by conferring resistance to various biostatic and biocidal drugs. In addition, a heterologous gene that is to be expressed in the cell also may advantageously be included within the construct. The arrangement might be as follows:
...vector~5'-flanking sequence~heterologous gene~selectable marker gene~flanking sequence-3'~vector...

Thus, using this kind of construct, it is possible, in a single recombinatorial event, to (i) "knock out" an endogenous gene, (ii) provide a selectable marker for identifying such an event and (iii) introduce a heterologous gene for expression.
5 Another refinement of the homologous recombination approach involves the use of a "negative" selectable marker. This marker, unli)';e the selectable marker, causes death of cells which express the marker. Thus, it is used to identify undesirable recombination events. When seeking to select homologous recombinants using a selectable marker, it is difficult in the initial screening step to identify proper homologous recombinants from recombinants generated from 10 random, non-sequence specific events. These recombinants also may contain the selectable marker gene and may express the heterologous protein of interest, but will, in all likelihood, not have the desired "knock out" phenotype. By attaching a negative selectable marker to the construct, but outside of the flanking regions, one can select against many random recombination events that will incorporate the negative selectable marker. Homologous recombination should 15 not introduce the negative selectable marker, as it is outside of the flanking sequences.
II. Transduction of cells The present invention provides improved compositions and methods for the transduction 20 of cells, particularly with viral vectors. In various aspects of the present invention, target cells (Section III below) can be transduced either in vitro, in vivo or ex vivo, depending on the particular application (Section VII, Subsection C below). As discussed in Section I above, the expression of (3-interferon can be inhibited, or the: activity of ~3-interferon can be neutralized in the target cells. In certain aspects of the invention, the inhibition of ~i-interferon is effected for a 25 defined and limited period of time, thereby limiting the effect that inhibition of ~3-interferon in cells may have.
A. Time frame of (3-interferon inhibition ~3-interferon is constitutively expressed in some cells, and can be transiently induced in 30 many types of cells. As discussed above, inhibition of (3-interferon expression or activity can lead to increased transduction efficiency. Studies conducted by the inventors indicate that a useful time frame for inhibition of (3-interferon e:Kpression and/or neutralization of (3-interferon activity, in order to increase the transduction efficiency, is while the cells interact with the virus.
In particular aspects of the present invention, the period of inhibition of (3-interferon expression or activity is between about 24 hours before to between about 24 to 48 hours after the administration of the viral vector.
S
B. Transduction with Viral Vectors In numerous aspects of the present invention, a heterologous gene is introduced into a host cell. For example, in the gene therapy aspects of the present invention, a construct comprising a therapeutic gene is delivered to cells in need of therapy.
Alternatively, in the aspects of the present invention involving production of selected proteins in vitro, an expression construct is delivered to an appropriate host cell. The present invention contemplates a variety of methods for the delivery of transducing constructs to host cells, as discussed in more detail below.
In certain aspects of gene therapy, the stability of the transducing construct is paramount to the success of the therapy regimen. The stability of the constructs can be effected in various ways. In certain embodiments of the invention, the nucleic acid encoding a selected gene may be stably integrated into the genome of the cell. In yet further embodiments. the nucleic ac;~i may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
1. Adenoviral Vectors A particular method for delivery of the expression constructs involves the use of an adenovirus expression vector. Although adenovirus vectors are known to have a low capacity for integration into genomic DNA, this feature is counterbalanced by the high efficiency of gene transfer afforded by these vectors. "Adenovirus expression vector" is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to ultimately express a tissue-specific transforming construct that has been cloned therein.

The expression vector comprises a genetically engineered form of adenovirus.
Knowledge of the genetic organization or adenovirus, a 36 kb, linear, double-stranded DNA
virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992). In contrast to re;trovirus, the adenoviral infection of host cells does not result in chromosomal integration becau:;e adenoviral DNA can replicate in an episomal manner without, potential genotoxicity. Also, adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification.
Adenovirus is particularly suitable for use; as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wiide target-cell range and high infectivity. Both ends of the viral genome contain 100-200 base; pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging. The early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA
replication. The E 1 region (E 1 A and E I B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A
and E2B) results in the synthesis of the proteins for viral DNA replication.
These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5'-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.
In a current system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provinls vector. Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process.
Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure.
Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses E I proteins (E 1 A and E 1 B; Graham et al. , 1977). Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the E 1, the D3 or both regions (Graham and Prevec, 1991 ). In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity for about 2 extra kb of DNA. Combined with the approximately 5.5 kb of DNA that is replaceable in the E l and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kb, or about 15% of the total length of the vector. More than 80%
of the adenovirus viral genome remains in the vector backbone.
Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.
Recently, Racher et al. ( 1995) disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by inoculating individual cells into I liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 rpm, the cell viability is estimated with trypan blue.
In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h. The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25%
of the final volume) and adenovirus added at an MOI of 0.05. Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.
Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus E 1 region. Thus, it will be most convenient to introduce the transforming construct at the position from which the E 1-coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted in lieu of the deleted E3 region in E3 replacement vectors as described by Karlsson et al. ( 1986) or in the E4 region where ;~ helper cell line or helper virus complements the E4 defect.
Adenovirus growth and manipulation is known to those of skill in the art, and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 109-1011 plaque-forming units per ml, and they are highly infective. The life cycle of adenovirus does not require integration into the host cell genome. The foreign genes delivered by adenovirus vectors are episomal and, therefore, have tow genotoxicity to host cells. No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al., 1963 ; Top et al. , 1971 ), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors.
Adenovirus vectors have been used in euli:aryotic gene expression (Levrero et al. , 1991;
Gomez-Foix et al., 1992) and vaccine development (Grunhaus and Horwitz, 1992;
Graham and Prevec, 1992). Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perric;~udet, 1991; Stratford-Perricaudet et al. , 1991;
Rich et al., 1993). Studies in administering recornbinant adenovirus to different tissues include trachea instillation (Rosenfeld et al., 1991; Rosenfeld et al., 1992), muscle injection (Ragot et al., 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al. , 1993 ). Recombinant adenovirus and adeno-associated virus (see below) can both infect and transduce non-dividing human primary cells.
2. AAV Vectors 5 Adeno-associated virus (AAV) is an attractive vector system for use in the cell transduction of the present invention as it has a high frequency of integration and it can infect nondividing cells, thus making it useful for delivery of genes into mammalian cells, for example, in tissue culture (Muzyczka, 1992) or in vivo. AAV has a broad host range for infectivity (Tratschin, et al., 1984; Laughlin, et al., 1986; Lebkowski, et al., 1988;
McLaughlin, et al., 10 1988). Details concerning the generation and use of rAAV vectors are described in U.S. Patent No. 5,139,941 and U.S. Patent No. 4,797,368, each incorporated herein by reference.
Studies demonstrating the use of AAV in gene delivery include LaFace et al. ( 1988);
Zhou et al. (1993); Flotte et al. (1993); and Walsh et al. (1994). Recombinant AAV vectors have 1 S been used successfully for in vitro and in vivo transduction of marker genes (Kaplitt, et al., 1994;
Lebkowski, et al., 1988; Samulski, et al., 1989; Shelling and Smith, 1994;
Yoder, et al., 1994;
Zhou, et al., 1994; Hermonat and Muzyczka, 1984; Tratschin, et al., 1985;
McLaughlin, et al., 1988) and genes involved in human diseases (Flotte, et al., 1992; Luo, et al., 1994; Ohi, et al., 1990; Walsh, et al., 1994; Wei, et al., 1994). Recently, an AAV vector has been approved for 20 phase I human trials for the treatment of cystic fibrosis.
AAV is a dependent parvovirus in that it requires coinfection with another virus (either adenovirus or a member of the herpes virus family) to undergo a productive infection in cultured cells (Muzyczka, 1992). In the absence of coinfection with helper virus, the wild type AAV
25 genome integrates through its ends into human chromosome 19 where it resides in a latent state as a provirus (Kotin et al., 1990; Samulski et al., 1991 ). rAAV, however, is not restricted to chromosome 19 for integration unless the AAV Rep protein is also expressed (Shelling and Smith, 1994). When a cell carrying an AAV provirus is superinfected with a helper virus, the AAV genome is "rescued" from the chromosome or from a recombinant plasmid, and a normal 30 productive infection is established (Samulski, et al. , 1989; McLaughlin, et al. , 1988; Kotin, et al., 1990; Muzyczka, 1992).

WO 98!22605 PCT/US97/21475 Typically, recombinant AAV (rAAV) virus is made by cotransfecting a plasmid containing the gene of interest flanked by the two AAV terminal repeats (McLaughlin et al., 1988; Samulski et al., 1989; each incorporated herein by reference) and an expression plasmid containing the wild type AAV coding sequences without the terminal repeats, for example pIM45 (McCarty et al., 1991; incorporated herein by reference). The cells are also infected or transfected with adenovirus or plasmids carrying the adenovirus genes required for AAV helper function. rAAV virus stocks made in such fashion are contaminated with adenovirus which must be physically separated from the rAAV particles (for example, by cesium chloride density centrifugation). Alternatively, adenovirus vectors containing the AAV coding regions or cell lines containing the AAV coding regions and some or all of the adenovirus helper genes could be used (Yang et al., 1994; Clark et al., 1995). Cell tines carrying the rAAV DNA
as an integrated provirus can also be used (Flotte et al., 1995).
3. Retroviral Vectors The retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in inff;cted cells by a process of reverse-transcription (Coffin, 1990). The resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins. The integration results in the retention of the viral gene sequences in the recipient cell and its descendants. The retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively. A sequence found upstream from the gag gene contains a signal for packaging of the genome into virions. Two long l;erminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome. These contain strong promoter and enhancer sequences and are also required for integration in the host celil genome (Coffin, 1990).
In order to construct a retroviral vector, .a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective. In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al., 1983). When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RN,A transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et al., 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer. Retroviral vectors are able to infect a broad variety of cell types. However, integration and stable S expression require the division of host cells (Paskind et al., 1975).
Concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication-competent virus in the packaging cells. This can result from recombination events in which the intact sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome. However, new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al., 1988; Hersdorffer et al., 1990).
4. Other viral vectors Other viral vectors may be employed as expression constructs in the present invention.
Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988), sindbis virus and herpesviruses may be employed.
They offer several attractive features for various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences. In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al. , 1990). This suggested that large portions of the genome could be replaced with foreign genetic material. Chang et al.
recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre-surface coding sequences. It was cotransfected with wild-type virus into an avian hepatoma cell line. Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes. Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al., 1991 ).

5. Modified Viruses In still further embodiments of the present invention, the nucleic acids to be delivered are housed within an infective virus that has been engineered to express a specific binding ligand.
The virus particle will thus bind specifically to the; cognate receptors of the target cell and deliver S the contents to the cell. A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification can permit the specific infection of hepatocytes via sialoglycoprotein receptors.
Another approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al., 1989). Using antibodies against major histocompatibility complex class I and class II
antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et .al., 1989).
C. Other types of DNA delivery to cells In certain aspects of the present invention, certain nucleic acid constructs need to be administered to cells prior to transduction. In these aspects, delivery of the constructs using viral vectors may be inappropriate. Therefore, several :non-viral methods for the transfer of expression constructs into cells also are contemplated by the: present invention. In one embodiment of the present invention, the expression construct may consist only of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned which physically or chemically permeabilize the cell membrane.
In certain embodiments of the present invention, the expression construct may be entrapped in a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991 ). Also contemplated is an expression construct complexed with Lipofectamine (Gibco BRL).
In certain embodiments of the invention, the liposome may be complexed with a S hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
In other embodiments of the present invention, the expression construct is introduced into the cell via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge.
In other methods of DNA delivery contemplated in the present invention, the expression construct is introduced to the cells using calcium phosphate precipitation. In another embodiment, the expression construct is delivered into the cell using DEAE-dextran followed by polyethylene glycol.
Another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment. This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al. , 1987). Further embodiments of the present invention include the introduction of the expression construct by direct microinjection or sonication loading.
In still other aspects of the present invention, the expression construct is introduced into the cell using adenovirus assisted transfection. Increased transfection efficiencies have been reported in cell systems using adenovirus coupled systems (Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994). Still further expression constructs that may be employed to deliver the construct to the target cells are receptor-mediated delivery vehicles.
These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in the target cells. In other embodiments, the DN,4 delivery vehicle component of a cell-specific gene targeting vehicle may comprise a specific binding ligand in combination with a liposome.
The nucleic acids to be delivered are housed within the liposome and the specific binding ligand is functionally incorporated into the iiposome membrane.

In still further embodiments, the DNA delivery vehicle component of the targeted delivery vehicles may be a liposome itself, which will preferably comprise one or more lipids or glycoproteins that direct cell-specific binding.
10 III. Cell Types The present invention discloses improved methods for treatment of genetically-based diseases (Section IV below), through more efficient transduction of therapeutic genes in target cells. Therefore, all cell types which can be targeted by gene therapy are contemplated for use in 15 the present invention. Additional aspects of the p~°esent invention concern the in vitro production of proteins (Section IX below). Different cell types may be required, depending on the particular protein being produced, the method of in vitro production, and the scale of production.
Exemplary, but not limiting, cell types contemplated for use in the present invention are discussed in more detail below.
A. Tumor Infiltrating Lymphocytes Tumor infiltrating lymphocytes are contemplated for use in the present invention, particularly in methods associated with the treatment of tumors. Tumor infiltrating lymphocytes (TIL) are lymphoid cells that infiltrate solid tumors. TILs are identified by immunohistochemical detection of CD3 or CD4/CD8 in tumor sections. TIL can be isolated from neoplasms by enzymatic dissociation, and then cultured in vitro with interleukin-2 (IL-2).
IL-2 stimulates the division of lymphoid cells, aJld, thus, the number of TILs can be expanded significantly (Topalian, 1995).
In some cases, TILs in tumors have been primed by tumor-associated antigens, such as breast and colon carcinomas (Vose and White, 1983), ovarian cancer (Ioannides et al., 1991 ) and melanoma (Topalian et al., 1989). In culture, TILs have been shown to mediate specific cytotoxicity against cancer cells (Topalian, 1995). The adoptive transfer of TIL by intravenous administration into mice (Topalian, 1995) or humans (Rosenberg, 1995) results in T1L homing to some cancer metastases.
TILs have also been transfected in vitro with TNF-oc genes. These genetically modified TILs also home to specific tumor sites (Rosenberg, 1995). In certain particular aspects of the present invention, TILs are transduced in vitro with a viral vector comprising an elastase gene and a GM-CSF gene, and administered to a cancer patient to produce angiostatin at tumor sites.
B. Macrophages One of the cell types contemplated for use in the present invention is macrophages.
Macrophages are specialized white blood cells which are descendent from a progenitor cell called the granulocyte/macrophage progenitor cell. Macrophages are involved in host defense by eliminating invading microorganisms through phagocytosis. Foreign particle recognition occurs through the binding of antibodies which react with the surface of the particle and are recognized by the Fc receptors on the surface of macrophages. Macrophages also scavenge senescent and damaged host cells. Macrophages play an essential role in homeostasis by participating in wound healing, chronic inflammatory reactions, tissue remodeling, and host defense against neoplasms (Fidler, 1985, 1994). In addition, macrophages are known to infiltrate certain types of tumors.
During many of these processes, macrophages degrade extracellular matrix proteins via secretion of matrix metalloproteinases (MMPs) that include interstitial collagenase, stromelysin, type IV collagenases (MMP-2 and MMP-9), and elastase (Belaaouaj et al., 1995;
Shapiro et al., 1993b; Xie et al., 1994). Although the MMPs share certain biochemical properties, each has a distinct substrate specificity (Matrisian, 1992). Which of these enzymes the macrophage produces depends on its level of differentiation and on tight regulation by many physiologic, pathologic, and pharmacological stimuli (Welgus et al., 1985; Busiek et al., 1992; Lacraz et al., 1992; Xie et al. , 1994).
The elastases can be divided into serine proteinases, which include pancreatic elastase (Shotton and Hartley, 1970) and neutrophil elastase (Ohlsson and Olsson, 1974), and the WO 98!22605 PCT/US97/21475 metalloelastase (MME) secreted by macrophages (White et al., 1977). The substrates for elastase include type I V collagen, immunoglobul.in, some glycoproteins such as a 1-proteinase inhibitor, and elastin, but not gelatin (Banda et al., 1980, 1983; Banda and Werb, 1981; Werb and Gordon, 1975). The genes coding for both human and murine MME have recently been cloned and characterized (Shapiro et al., 1992, :1993a). MME is essential for penetration of basement membranes and tissue invasion by macrophages (Shipley et al., 1996a, b), which can occupy up to 60% of the tumor mass (Bucana et al. , 1992; Mantovani et al. , 1992; Normann, 1985; Whitworth et al., 1990). Since angiostatin can be generated from plasminogen by pancreatic elastase in vitro, (O'Reilly et al. , 1994), the inventors reasoned that tumor-infiltrating macrophages could be induced to express MME, which in turn would generate angiostatin in vivo. This is shown to be the case in the instant disclosure (Examples 10 and 11 below).
C. Bone Marrow Macrophages Particularly preferred for use in the present invention are bone marrow macrophages.
1 S Bone marrow macrophages are collected from aspirates, and grown in suspension cultures to derive a large number of cells which can be transduced using the methods of the instant invention. These cells can then be administered. to a patient and home to cancer metastases, whereupon they can produce angiostatin.
D. Bone Marrow Celts Another type of cells contemplated for use in the present invention are bone marrow cells. Bone marrow cells can be readily collected from patients and grown in culture.
Transduction of these cells in vitro with viral and non-viral vectors has been described, albeit at low efficiencies (Deisseroth, 1993; Deisseroth et al., 1994; Hanania and Deisseroth, 1994).
Using the methods disclosed herein, bone marrow cells can be efficiently transduced with a selected gene or genes, and the genetically modified cells can then be injected directly into tumors or into the circulation.
E. Tumor Cells Another particular type of cells for use in the present invention are tumor cells.
Replicating tumor cells or tumor cells that do not have invasive or metastatic potential can be used in the present invention. For example, and not limitation, tumor cells transduced to produce GM-CSF can be lethally X-irradiated, and reintroduced into a tumor. These cells will not divide, yet will remain metabolically active for several days. Noninvasive-nonmetastatic tumor cells will only grow at the local site. Cells transduced with the GM-CSF gene will recruit and activate macrophages to produce angiostatin.
F. Endothelial Cells Also contemplated for use in the present invention are endothelial cells.
Endothelial cells line all blood vessels and lymphatics. Endothelial cells are the key cellular component of all blood vessels, and are stimulated to produce new blood vessels in wound healing, tissue repair and regeneration and in neoplasms, i.e., the process of angiogenesis.
G. Epithelial Cells Another particular type of cells for use in the present invention are epithelial cells.
Epithelial cells are found throughout the body, including the skin, the linings of the oral cavity, the digestive tract, the urogenital system and the respiratory tract.
Epithelial cells can be readily cultivated in culture, and serve as recipients for transduction.
H. Fibroblasts Fibroblasts are the supporting structural cells of every organ in the body.
Skin fibrobiasts can be readily cultivated in culture and can thus be transduced by the methods of the present invention with a variety of genes (Section VI below).
IV. Diseases There are a number of diseases which are genetically-based, and therefore amenable to treatment by the improved methods and compositions for gene therapy disclosed in the present invention. These include, but are not limited to, cancer, angiogenesis related diseases, viral infections, including AIDS, and diseases caused by a missing or malfuctioning gene, such as ADA deficiency and cystic fibrosis.

A. Cancer Treatments for cancer are disclosed in the present invention. Since all neoplasms require an adequate blood supply for growth (angiogenesis), all neoplasms are amenable to antiangiogenic therapy, in particular, the GM-CSF stimulated elastase mediated production of angiostatin. This therapy can be directed against primary neoplasms and disseminated cancer metastases located in different body organs, e.~;., lung, liver, bone and brain. Exemplary neoplasms are prostate, breast, melanoma, colon, F~ancreas and lung.
In a particular embodiment of the present invention, bone marrow macrophages are isolated from aspirates, and grown in suspension cultures. A transducing composition comprising a gene encoding GM-CSF and a ~3-inte.rferon inhibitory factor is then administered to the macrophage culture. The transduced macrophages are then administered ex vivo to a patient in need of cancer therapy by any of the routes described herein, but preferably intravenously or intratumorally. The macrophages then naturally home to the sites of tumors, both primary and metastatic, whereupon the transduced GM-CSF gene induces the production of MME. The MME is secreted from the macrophages, and converts endogenous plasminogen to angiostatin at the tumor site, thus preventing tumor growth by blocking the formation of new blood vessels.
In other aspects of the present invention, treatment of cancer using gene therapy may be combined with more conventional therapies, such ;~s chemotherapy (Section VIII
below).
B. Angiogenesis Related Diseases Treatments for other angiogenesis related diseases are also contemplated in the present invention. Inhibition of excessive blood vessel formation is useful for treatment of angiogenic diseases, such as collateral blood vessels, vascular restenosis, ocular neovascularization, infantile hemangioma, diabetic retinopathy, arthritis, psoriasis, endometriosis, duodenal ulcers (Folkman and Shing, 1992; Folkman, 1995) and pulmonary hypertension (Tuder et al., 1994).
C. Other Diseases Treated by Gene ~Cherapy Since the methods described in the present invention can increase transduction efficiency, any disease which can be treated by gene therapy is contemplated for treatment by the methods of the present invention. Examples of these diseases are vascular proliferative diseases (Gunman, 1994; Chang et al., 1995), hemophilia (Kay et al., 1994; Fang et al., 1995), a,l-antitrypsin deficiency (Rosenfeld et al., 1991 ), ornithine transcarbamoylase deficiency (Stratford-Perricaudet et al., 1991), muscle degeneration diseases (Stratford-Perricaudet et al., 1992), cystic fibrosis (Rosenfeid et al., 1992; Knowles et al., 1995), familial 5 hypercholesterolemia (reviewed by Crystal, 1995), ADA deficiency (Anderson, 1984), anemias and chronic infections.
V. Promoters and Enhancers 10 In certain aspects of the present invention, vectors which are designed for the expression of a desired gene or genes are required. Thus, particular embodiments may require a selected nucleic acid segment, for example, antisense constructs (Section I, B), viral constructs (Section II, B) and heterologous genes to be expressed in host cells (Section VI) to be operatively positioned relative to control sequences, such as promoters and enhancers.
Below are a list of 15 viral promoters, cellular promoterslenhancers and inducible promoters/enhancers that could be used in combination with the present invention. Additionally any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base, EPDB) could also be used to drive expression of exemplary structural genes encoding (3-interferon or (3-interferon related proteins such as IRF-1, elastase, GM-CSF, selectable marker proteins or a heterologous protein of interest 20 (Section VI below).

ENHANCER REFERENCES

Immunoglobulin Heavy ChainBanerji et al., 1983; Gilles et al., 1983; Grosschedl and Baltimore, 1985; Atchinson and Perry, 1986, 1987; Imler et al., 1987; Weinberger et al., 1984;

Kiledjian et al., 1988; Porton et al.;

Immunoglobulin Light ChainQueen and Baltimore, 1983; Picard and Schaffner, Table 1 (Continued) ENHANCER REFERENCES

T-Cell Receptor Luria et al., 1987; Winoto and Baltimore, 1989;
Redondo et al. ; 1990 HLA DQ a and DQ (3 Sullivan and :Peterlin, 1987 (3-Interferon Goodbourn et al., 1986; Fujita et al., 1987;
Goodbourn and Maniatis, 1988 Interleukin-2 Greene et al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin et al., 1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Sherman et al., 1989 ~i-Actin Kawamoto et al., 1988; Ng et al.;

Muscle Creatine Kinase Jaynes et al., 1988; Horlick and Benfield, 1989;
Johnson et al., 1989a I

Prealbumin (Transthyretin)Costa et al., 1988 Elastase I Omitz et al., '1987 Metallothionein Karin et al., 1987; Culotta and Hamer, Collagenase Pinkert et al., 1987; Angel et al., Albumin Gene Pinkert et al., 1987; Tronche et al., 1989, 1990 oc-Fetoprotein Godbout et al'., 1988; Campere and Tilghman, 1989 t-Globin Bodine and Ley, 1987; Perez-Stable and Constantini, ~i-Globin Trudel and Constantini, 1987 e-fos Cohen et al., 1987 c-HA-ras Triesman, 1986; Deschamps et al., ~ 1985 ' Insulin Edlund et al., 1985 Neural Cell Adhesion MoleculeHirsh et al., 1990 (NCAM) al-Antitrypain Latimer et al., 1990 Table 1 (Continued) ENHANCER REFERENCES

H2B (TH2B) Histone Hwang et al., 1990 Mouse or Type I Collagen Ripe et al., 1989 Glucose-Regulated ProteinsChang et al., 1989 (GRP94 and GRP78) Rat Growth Hormone Larsen et al., 1986 Human Serum Amyloid A Edbrooke et al., 1989 (SAA) Troponin I (TN I) Yutzey et al., 1989 Platelet-Derived Growth Pech et al., 1989 Factor Duchenne Muscular DystrophyKlamut et al., 1990 SV40 Banerji et al., 1981; Moreau et al., 1981; Sleigh and Lockett, 1985; Firak and Subramanian, 1986; Herr and Clarke, 1986; Imbra and Karin, 1986; Kadesch and Berg, 1986; Wang and Calame, 1986;
Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al., 1988 Polyoma Swartzendruber and Lehman, 1975; Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al., 1983; de Villiers et al., 1984;

Hen et al., 1986; Satake et al., 1988;
Campbell and Villarreal, 1988 Retroviruses Kriegler and Botchan, 1982, 1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b, 1988; Bosze et al., 1986; Miksicek et al., 1986;
Celander and Haseltine, 1987; Thiesen et al., 1988;
Celander et al., 1988; Choi et al., 1988; Reisman and Rotter, Table 1 (Continued) ENHANCER REFERENCES

Papilloma Virus Campo et al., 1983; Lusky et al., 1983;
Spandidos I and Wilkie, 1983; Spalholz et al., 1985; Lusky and Botchan, 1986; Cripe et al., 1987;
Gloss et al., 1987;

Hirochika et ~xl., 1987; Stephens and Hentschel, 1987; Glue et al., 1988 Hepatitis B Virus Bulla and Siddiqui, 1986; Jameel and Siddiqui, 1986; Shaul zu~d Ben-Levy, 1987; Spandau and Lee, 1988; Vannice and Levinson, 1988 Human Immunodef ciency Muesing et a!., 1987; Hauber and Cullan, Virus 1988;

Jakobovits et al., 1988; Feng and Holland, 1988;

Takebe et al.. 1988; Rosen et al., ~i 1988; Berkhout et al., 1989; I,aspia et al., 1989;
Sharp and Marciniak, 1 ~>89; Braddock et al., Cytomegalovirus Weber et al., 1984; Boshart et al., 1985; Foecking and Hofstetter, 1986 Gibbon Ape Leukemia VirusHolbrook et crl., 1987; Quinn et al., Element Inducer References MT II Phorbol Ester (TFA,) Palmiter et al., 1982;

Heavy metals Haslinger and Karin, 1985;

Searie et al., 1985;
Stuart et al., 1985; Imagawa et al., 1987, Karin et al., 1987;

Angel et al., 1987b;

McNeall et al., 1989 Table 2 (Continued) Element Inducer References MMTV (mouse mammary Glucocorticoids Huang et al., 1981;
tumor virus) Lee et al. , 1981; Majors and Varmus, 1983; Chandler et al., 1983; Lee et al., 1984; Ponta et al., 1985;
Sakai et al., 1988 (3-Interferon poly(rI)x Tavernier et al., poly(rc) 1983 Adenovirus 5 E2 Ela Imperiale and N evins, 1984 Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin Phorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA) Angel et al., 1987b Murine MX Gene Interferon, Newcastle Disease Virus GRP78 Gene A23187 Resendez et al., 1988 a,-2-Macroglobulin IL-6 Kunz et al., 1989 Vimentin Serum Rittling et al., 1989 MHC Class I Gene H-2KbInterferon Blanar et al., 1989 HSP70 Ela, SV40 Large T Taylor et al., 1989;
Antigen Taylor and Kingston, 1990a, b Proliferin Phorbol Ester-TPA Mordacq and Linzer, Tumor Necrosis FactorFMA Hensel et al., 1989 Thyroid Stimulating Thyroid Hormone Chatterjee et al., Hormone a Gene 1989 VI. Genes A number of different genes are contemplated for use in the present invention.
Among these are genes useful in treating diseases such as cancer, other angiogenesis related diseases, and SS
additional diseases which can be treated by gene therapy (Section IV above).
Additionally, marker genes used in the identification of recombinant cells are contemplated for use, as are genes encoding proteins to be produced by cells ir,! vitro.
A. GM-CSF
GM-CSF is a glycoprotein which stimulates the production of granulocytes and macrophages from the common granulocyte/macrophage progenitor cells. Due to the varying degree of glycosylation, the molecular weight has been reported to be between 14.5 and 35 kDa.
The protein consists of a single polypeptide chain of 127 amino acids. GM-CSF
has been cloned and sequenced from human (GenBank accession. number M 11220), gibbon, mouse (GenBank accession number X03019), cattle and sheep, as vvell as a partial sequence from rat. The details of the post receptor signal transduction pathway modulated by GM-CSF are largely unknown.
While GM-CSF treatment leads to the phosphorylation of tyrosine residues on a number of different proteins, the GM-CSF receptor has no kinase domain or any known signaling sequences.
I n certain aspects of the present invention, the GM-C S F gene is introduced into macrophages to stimulate MME expression, and thereby the production of angiostatin. In other embodiments, the GM-CSF gene is used to direcvt the production of GM-CSF in vitro, which is then used for systemic administration to stimulate MME production in macrophages which have infiltrated tumors.
B. Elastase The elastases can be divided into serine proteinases and metalloproteinases.
The serine proteinases include pancreatic elastase (Shotton and Hartley, 1970) and neutrophil elastase (Ohlsson and Olsson, 1974), and the metalloprotE;inases include the metalloelastase secreted by macrophages (MME; White et al., 1977). Metalloproteinases are involved in such functions as tissue remodeling, wound repair and embryonic development.
The macrophage related metalloelastase (MME) from mouse and human (GenBank accession number M82831 ) have been isolated and sequenced. In certain embodiments of the present invention, the MME gene is transduced, either alone or in combination with the GM-CSF

gene, into cells which are then administered to a tumor, to effect the in vivo production of angiostatin.
C. (3-interferon (3-interferon (IFN-Vii) is low molecular weight protein that is produced by many cell types, including epithelial cells, fibroblasts and macrophages (Sen and Lengyel, 1992). Cells that express endogenous IFN-(3 are resistant to viral infection and replication. The (3-interferon genes from mouse (GenBank accession numbers X 14455, X 14029) and human (GenBank accession numbers J00218, K00616 and M 11029) have been isolated and sequenced.
In particular embodiments of the present invention, the gene for ~i-interferon is used in an antisense construct, which is administeredto target cells to reduce the endogenous expression of (3-interferon, thereby effecting increased transduction efficiency.
D. IRF-1 and IRF-2 Two genes which are involved in the regulation of (3-interferon are termed interferon regulatory factor-I (IRF-I) and interferon regulatory factor-2 (IRF-2). IRF-1 is a positive control factor which binds to regulatory motifs present in the upstream region of interferon genes (Miyamoto et al., 1988). IRF-1 has been cloned from mouse (GenBank accession numbers M21065, M25560 and J03160) and human (GenBank accession number X 14454). IRF-2 is an antagonistic repressor of ~3-interferon expression (Harada et al. , 1989). IRF-2 has been cloned from mouse (GenBank accession number J03168) and human (GenBank accession number X15949).
In certain aspects of the present invention, the gene for IRF-1 is comprised within an antisense construct and administered to a cell targeted for transduction, thereby reducing the endogenous expression of (3-interferon and effecting efficient transduction of the target cell. In other aspects, the gene for IRF-2 is subcloned into an expression vector which is administered to cells targeted for transduction, thereby reducing the endogenous expression of (3-interferon and effecting efficient transduction of the target cell.

E. Heterologous Genes The present invention discloses methods for the efficient transduction of cells with a selected heterologous gene. The heterologous genes can be used, for example, in therapeutic applications, or for the in vitro production of a desired protein. Below is a list of selected cloned structural genes that could be used in the present invention. The list is not in any way meant to be interpreted as limiting, only as exemplary of ohe types of structural genes contemplated for use in the present invention.

Selected Cloned Structural Genes Gene Clone Type* Reference activin porcine-cDNA Mason AJ, Nat, 318:659, 1985 adenosine deaminaseh-cDNA Wiginton DA, PNAS, 80:7481, angiotensinogen r-cDNA Ohkubo H, PNAS, 80:2196, r-gDNA Tanaka T, JBC, 259:8063, antithrombin III H-cDNA Bock SC, NAR 10:8113, 1982 h-cDNA and gDNA Prochownik EV, JBC, 258:8389, antitrypsin, alphah-cDNA Karachi K, PNAS, 78:6826, h-gDNA Leicht M, Nat, 297:655, 1982 RFLP Cox DW, AJHG, 36:1345, 1984 apolipoprotein h-cDNA, h-gDNA Shoulders CC, NAR, 10:4873, RFLP Karathanasis SK, Nat, 301:718, h-gDNA Kranthanasis SK, PNAS, 80:6147, apolipoprotein h-cDNA Sharpe CR, NAR, 12:3917, Chr Sakaguchi, AY, AJHB, 36:2075, h-cDNA Knott TJ, BBRC, 120:734, Table 3 (Continued) Selected Cloned Structural Genes Gene Clone Type* Reference apolipoprotein h-cDNA Knott TJ, NAR, 12:3909, 1984 C-I

apolipoprotein h-cDNA Jackson CL, PNAS, 81:2945, h-cDNA Mykelbost O, JBC, 249:4401, h-cDNA Fojo SS, PNAS, 81:6354, 1984 RFLP Humphries SE, C Gen, 26:389, apolipoprotein h-cDNA and gDNA Karanthanasis SK, Nat, 304:371, h-cDNA Sharpe CR, NAR, 12:3917, apolipoprotein h-cDNA Brewslow JL, JBC, 257:14639, atrial natriuretich-cDNA Oikawa S, Nat, 309:724, 1984 factor h-cDNA Nakayama K, Nat, 310:699, h-cDNA Zivin RA, PNAS, 81:6325, h-gDNA Seidman CE, Sci, 226:1206, h-gDNA Nemer M, Nat, 312:654, 1984 h-gDNA Greenberg BI, Nat, 312:665, chorionic h-cDNA Fiddes JC, Nat, 281:351, gonadotropin, alpha chain RFLP Boethby M, JBC, 256:5121, chorionic h-cDNA Fiddes JC, Nat, 286:684, gonadotropin, beta chain h-gDNA Boorstein WR, Nat, 300:419, h-gDNA Talmadge K, Nat, 307:37, chymosin, pro (rennin)bovine-cDNA Harris TJR, NAR, 10:2177, complement, factorh-cDNA Woods DE, PNAS, 79:5661, h-cDNA and gDNA Duncan R, PNAS, 80:4464, complement C2 h-cDNA Bentley DR, PNAS, 81:1212, h-gDNA (C2, C4, Carroll MC, Nat, 307:237, and 1984 B) Table 3 (Continued) Selected Cloned Structural Genes Gene Clone Type* Reference I

complement C3 m-cDNA Domdey H, PNAS, 79:7619, h-gDNA 1983 Whitehead AS, PNAS, 79:5021, 1982 ~~

complement C4 h-cDNA and gDNA Carroll MC, PNAS, 80:264, h-cDNA 1983 Whitehead AS, PNAS, 80:5387, complement C9 h-cDNA DiScipio RC, PNAS, 81:7298, corticotropin releasingsheep-cDNA Furutani Y, Nat, 301:537, factor h-gDNA 1983 Shibahara S, EMBO J, 2:775, epidermal growth m-cDNA Gray A, Nat, 303:722, 1983 factor m-cDNA Scott J, Sci, 21:236, 1983 h-gDNA Brissenden JE, Nat, 310:781, epidermal growth h-cDNA and Chr Lan CR, Sci, 224:843, 1984 factor receptor,oncogene c-erb B

epoxide dehydrataser-cDNA ~Gonzlalez FJ, JBC, 256:4697, erythropoietin h-cDNA Lee-Huang S, PNAS, 81:2708, esterase inhibitor,h-cDNA 'Stanley KK, EMBO J, 3:1429, Cl 1984 -factor VIII h-cDNA and gDNA Gitschier J, Nat, 312:326, h-cDNA 1984 'Toole JJ, Nat, 312:342, factor IX, Christmash-cDNA Kutachi K, PNAS, 79:6461, factor h-cDNA 1982 RFLP Choo KH, Nat, 299:178, 1982 h-gDNA Camerino G, PNAS, 81:498, .Elnson DS, EMBO J, 3:1053, factor X h-cDNA :Leytus SP, PNAS, 81:3699, fibrinogen A alpha,h-cDNA ;Kant JA, PNAS, 80:3953, Table 3 (Continued) Selected Cloned Structural Genes Gene Clone Type* Reference B beta, gamma h-gDNA (gamma) Fornace AJ, Sci, 224:161, h-cDNA (alpha Imam AMA, NAR, I 1:7427, gamma) Fornace AJ, JBC, 259:12826, h-gDNA (gamma) gastrin releasing h-cDNA Spindel ER, PNAS, 81:5699, peptide glucagon, prepro hamster c-DNA Bell GI, Nat, 302:716, 1983 h-gDNA Bell GI, Nat, 304:368, 1983 growth hormone h-cDNA Martial JA, Sci, 205:602, h-gDNA DeNoto FM, NAR, 9:3719, 1981 GH-like gene Owerbach, D, Sci, 209:289, growth hormone, h-cDNA Gubler V, PNAS, 80:341 I, RF, 1983 somatocrinin h-cDNA Mayo KE, Nat, 306:86:1983 hemopexin h-cDNA Stanley KK, EMBO J, 3:1429, inhibin porcine-cDNA Mason AJ, Nat, 318:659, 1985 insulin, prepro h-gDNA Ullrich a, Sci, 209:612, insulin-like growthh-cDNA Jansen M, Nat, 306:609, 1983 factor I h-cDNA Bell GI, Nat, 310:775, 1984 Chr Brissenden JE, Nat, 310:781, insulin-like growthh-cDNA Bell GI, Nat, 310:775, 1984 factor II h-gDNA Dull TJ, Nat, 310:777, 1984 Chr Brissenden JE, Nat, 310:781, interferon, alpha h-cDNA Maeda S, PNAS, 77:7010, 1980 (leukocyte), multipleh-cDNA (8 distinct)Goeddel DV, nat, 290:20, I h-gDNA Lawn RM, PNAS, 78:5435, 1981 h-gDNA Todokoro K, EMBO J, 3:1809, h-gDNA Torczynski RM, PNAS, 81:6451, WO 98/22605 PCT/(TS97/21475 Table 3 (Continued) Selected Cloned Structural Genes Gene Clone Type* Reference interferon, beta h-cDNA Taniguchi T, Gene, 10:1 l, (fibroblast) h-gDNA 1980 h-gDNA (related) Lawn RM, NAR, 9:1045, 1981 h-gDNA (related) Sehgal P, PNAS, 80:3632, Sagar AD, Sci, 223:1312, interferon, gamma h-cDNA Gray PW, Nat, 295:503, 1982 (immune) h-gDNA Gray PW, Nat, 298:859, 1982 interleukin-1 m-cDNA Lomedico PT, Nat, 312:458, interleukin-2, h-cDNA Devos R, NAR, 11:4307, 1983 T-cell growth factor h-cDNA Taniguchi T, Nat, 302:305, h-gDNA 1983 Chr Hollbrook NJ, PNAS, 81:1634, Siegel LF, Sci, 223:175, interleukin-3 m-cDNA Fung MC, Nat, 307:233, 1984 kininogen, two bovine-cDNA Nawa H, PNAS, 80:90, 1983 forms bovine,-cDNA and Kitamura N, Nat, 305:545, gDNA 1983 leuteinizing hormone,h-gDNA and Chr Talmadge K, Nat, 207:37, beta subunit 1984 leuteinizing hormoneh-cDNA and gDNA Seeburg PH, Nat, 311:666, releasing hormone lymphotoxin h-cDNA and gDNA Gray PW, Nat, 312:721, 1984 mast cell growth m-cDNA Yokoya T, PNAS, 81:1070, factor 1984 nerve growth factor,m-cDNA Scott J, Nat, 302:538, 1983 beta subunit h-gDNA Ullrich A, Nat, 303:821, Chr 1983 Franke C, Sci, 222:1248, oncogene, c-sis, h-gDNA Dalla-Favera R, Nat, 295:31, chain A h-cDNA Clarke MF, Nat, 208:464, Table 3 (Continued) Selected Cloned Structural Genes Gene Clone Type* Reference pancreatic polypeptideh-cDNA Boel E, EMBO J, 3:909, 1984 and icosapeptide parathyroid hormone,h-cDNA Hendy GN, PNAS, 78:7365, prepro h-gDNA Vasicek TJ, PNAS, 80:2127, plasminogen h-cDNA and gDNA Malinowski DP, Fed P, 42:1761, plasminogen activatorh-cDNA Edlund T, PNAS, 80:349, 1983 h-cDNA Pennica D, Nat, 301:214, h-gDNA Ny T, PNAS, 81:5355, 1984 prolactin h-cDNA Cook NE, JBC, 256:4007, 1981 r-gDNA Cooke NE, Nat, 297:603, 1982 proopiomelanocortinh-cDNA DeBold CR, Sci, 220:721, h-gDNA Cochet M, Nat, 297:335, 1982 protein C h-cDNA Foster D, PNAS, 81:4766, prothrombin bovine-cDNA MacGillivray RTA, PNAS, 77:5153, relaxin h-gDNA Hudson P, Nat, 301:628, 1983 h-cDNA (2 genes) Hudson P, EMBO J, 3:2333, Crawford, RJ, EMBO J, 3:2341, renin, prepro h-cDNA Imai T, PNAS, 80:7405, 1983 h-gDNA Hobart PM, PNAS 81:5026, h-gDNA Miyazaki H, PNAS, 81:5999, Chirgwin 3M, SCMG, 10:415, somatostatin h-cDNA Shen IP, PNAS, 79:4575, 1982 h-gDNA and Ri-IP Naylot SI, PNAS, 80:2686, tachykinin, prepro,bovine-cDNA Nawa H, Nat, 306:32, 1983 substances P & bovine-gDNA Nawa H, Nat, 312:729, 1984 K

WO 98/22605 PCTlUS97/21475 Table 3 (Continued) Selected I~loned Structural Genes ~

Gene Clone Type* Reference urokinase h-cDNA Verde P, PNAS, 81:4727, 1984 vasoactive intestinalh-cDNA Itoh N, Nat, 304:547, 1983 peptide, prepro vasopressin r-cDNA Schmale H, EMBO J, 2:763, Key to Table 3: *cDNA - complementary DNA; Chr - chromosome; gDNA - genomic DNA;
RFLP - restriction fragment polymorphism; h - hu:man; m - mouse; r - rat F. Oncogenes and Mutant Tumor Suppressors In certain aspects of the present invention, antisense constructs to oncogenes are used in methods of treating cancer. In other aspects, transformation of target cells is desired. Exemplary transforming genes and constructs are listed below. These genes fall into different functional categories, such as those that perturb signal transduction, affect cell cycle, alter nuclear transcription, alter telomere structure or function, inhibit apoptosis, or that exert pleiotropic activities. It will be understood that the genes listed are only exemplary of the types of oncogenes, mutated tumor suppressors and other transforming genetic constructs and elements that may be used in this invention. Further transfbrming genes and constructs will be known to those of ordinary skill in the art.
A number of proteins have been shown to inhibit apoptosis, or programmed cell death.
Representative of this class are bcl-2 (distinct from bcl-1, cyclin D 1;
GenBank Accession No.
M14745, X06487) and family members including Bcl-xl, Mci-1, Bak, A1, A20, and inhibitors of interleukin-1 (3-converting enzyme and family members. Overexpression of this oncogene was first discovered in T cell lymphomas. It functions as an oncogene by binding and inactivating bax, a protein in the apoptotic pathway.
In addition to proteins which inhibit apoptosis, a large number of proteins have been reported which fail to promote apoptosis. Among these are p53, retinoblastoma gene (Rb), Wilm's tumor ( WT 1 ), bax alpha, interleukin-1 (3-converting enzyme and family, MEN-1 gene {chromosome 11 q 13 ), neurofibromatosi s, type 1 (NF 1 ), cdk inhibitor p 16, p21, colorectal cancer gene (DCC), familial adenomatosis polyposis gene (FAP), multiple tumor suppressor gene (MTS-1), BRCA1, BRCA2.
S
Preferred are p53 and the retinoblastoma gene. Most forms of cancer have reports of p53 mutations. Inactivation of p53 results in a failure to promote apoptosis. With this failure, cancer cells progress in tumorigenesis rather than be destined for cell death. A
short list of cancers and mutations found in p53 is: ovarian (GenBank Accession No. 553545, 562213, S62216); liver (GenBank Accession No. 562711, 562713, S62714, S6771 S, 572716); gastric (GenBank Accession No. 563157); colon (GenBank Accession No. S63610); bladder (GenBank Accession No. S85568, 585570, 585691 ); lung (GenBank Accession No. 541969, S41977);
glioma (GenBank Accession No. S85807, 585712, S85713).
1 S There are a number of known oncogenes and mutant tumor suppressors which act by perturbing signal transduction. Representative members of this class are tyrosine kinases, both cytoplasmic and membrane-associated forms, such as the Src family, JaklStats, Ros, Neu, Fms, Ret, Abl and Met. Other members of this class are serine/threonine kinases, such as Mos, Raf, protein kinase C (PKC) and PIM-1. Another family of proteins which fall into this class are the growth factors and receptors, such as platelet derived growth factor (PDGF), insulin-like growth factor (IGF-1 ), insulin receptor substrate (IRS-1 and IRS-2), the Erb family, epidermal growth factor (EGF), growth hormone, hepatocyte growth factor (HGF) basic fibroblast growth factor (bFGF), as well as the corresponding growth factor receptors. Small GTPases or G proteins also belong to this class, and are represented by the ras family, rab family, and Gs-alpha. Receptor-type tyrosine phosphatase IA-2 is also a member of this class of proteins.
Exemplary of the members contemplated for use in the present invention are Neu, also known as Her2, also known as erbB-2 (GenBank accession numbers M11730, X03363, and S57296). Discovered as an oncogene in breast cancer, found also in other forms of cancer as well. This seems to be a member of the receptor tyrosine kinase family. Also preferred is hepatocyte growth factor receptor (HGFr; GenBank accession number U11813), also known as scatter factor receptor. This can be an example of a receptor, either endogenously present or expressed from a recombinant adenovirus, that is used to stimulate proliferation of a target cell population. Other particular members are insulin-like growth factor 1 receptor (GenBank accession number X04434 and M24599), and G'TPase Gs alpha (GenBank accession numbers X56009, X04409). Gs alpha is associated with pituitary tumors that secrete growth hormone, but 5 not other neuroendocrine or endocrine tumors.
Transforming genes have also been described which affect the cell cycle.
Proteins which belong to this class are the cyclin-dependent protean kinases (cdk), classes A-E; and members of the cyclin family such as cyclin D. Exemplary for use in the present invention is cyclin D1, also 10 known as PRAD, also known as bcl-1 (GenBar.~k accession numbers M64349 and M73554).
This is associated as an oncogene primarily with parathyroid tumors.
A number of transforming genes have been described which assert their effect through an alteration of nuclear transcription. This class includes the Myc family members including c-myc, 15 N-myc, and L-myc; the Rel family members including NF-KB; c-Myb, Ap-l, fos, and jun, insulinoma associated cDNA (IA-1), Erbb-1, and the PAX gene family. Exemplary for use in the present invention is c-myc (GenBank accession numbers J00120, K01980, M23541, V00501, X00364.
20 A protein which has recently been implicated in cellular transformation is telomerase.
Telomerase is involved in the assembly and maintenance of telomeres, which are at the end of chromosomes. It is presently unknown how telomerase functions as in transformation.
Some transforming genes have pleiotropic effects. Among these proteins are viral 25 proteins such as SV40 and polyoma large T antigens, SV40 temperature sensitive large T
antigen, adenovirus E 1 A and E 1 B proteins, and papillomavirus E6 and E7 proteins. Selected from this class is SV40 large T antigen (TAG; Ger~Bank accession number J02400).
G. Marker genes 30 In certain aspects of the present invention, specific cells are tagged with specific genetic markers to provide information about the fate of the tagged cells. Therefore, the present invention also provides recombinant candidate screening and selection methods which are based WO 98/22605 PCTlUS97/21475 upon whole cell assays and which, preferably, employ a reporter gene that confers on its recombinant hosts a readily detectable phenotype that emerges only under conditions where a general DNA promoter positioned upstream of the reporter gene is functional.
Generally, reporter genes encode a polypeptide (marker protein) not otherwise produced by the host cell which is detectable by analysis of the cell culture, e.g., by fluorometric, radioisotopic or spectrophotometric analysis of the cell culture.
In other aspects of the present invention, a genetic marker is provided which is detectable by standard genetic analysis techniques, such as DNA amplification by PCRT""
or hybridization using fluorometric, radioisotopic or spectrophotometric probes.
1. Screening Exemplary enzymes include esterases, phosphatases, proteases (tissue plasminogen activator or urokinase) and other enzymes capable of being detected by their activity, as will be known to those skilled in the art. Contemplated for use in the present invention is green fluorescent protein (GFP) as a marker for transgene expression (Chalfie et al., 1994). The use of GFP does not need exogenously added substrates, only irradiation by near UV or blue light, and thus has significant potential for use in monitoring gene expression in living cells.
Other particular examples are the enzyme chloramphenicol acetyltransferase (CAT) which may be employed with a radiolabelled substrate, firefly and bacterial luciferase, and the bacterial enzymes (3-galactosidase and (3-glucuronidase. Other marker genes within this class are well known to those of skill in the art, and are suitable fox use in the present invention.
2. Selection Another class of reporter genes which confer detectable characteristics on a host cell are those which encode polypeptides, generally enzymes, which render their transformants resistant against toxins. Examples of this class of reporter genes are the neo gene (Colberre-Garapin et al., 1981 ) which protects host cells against toxic levels of the antibiotic 6418, the gene conferring streptomycin resistance (IJ. S. Patent 4,430,434), the gene conferring hygromycin B
resistance (Santerre et al., 1984; U. S. Patents 4,727,028, 4,960,704 and 4,559,302), a gene encoding dihydrofolate reductase, which confers resistance to methotrexate (Alt et al., 1978), the enzyme HPRT, along with many others well kno~m in the art (Kaufman, 1990).
VII. Pharmaceutically Acceptable Compositia~ns and Routes of Administration The present invention discloses numerous compositions, which in certain aspects of the invention, are administered to animals. For example, the instant invention discloses nucleic acids, both DNA, such as genes encoding (3-interferon or GM-CSF, and RNA, such as anitsense and ribozymes, proteins such as GM-CSF, peptides such as ~i-interferon-related kinase inhibitors, antibodies such as anti ~i-interferon polyclonal or monoclonal antibodies, viruses such as adenoviruses or retroviruses and chemicals such as ~3-interferon-related kinase inhibitors and chemotherapeutics for use in animals.
Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions of the viruses and cells in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
One will generally desire to employ appropriate salts and buffers to render viruses or cells suitable for introduction into a patient. Aqueous compositions of the present invention comprise an effective amount of viruses or cells, dissolved or dispersed in a pharmaceutically acceptable earner or aqueous medium, and preferably encapsulated. The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agent,, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well know in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated.
Supplementary active ingredients, such as other anti-cancer agents, can also be incorporated into the compositions.

WO 98!22605 PCT/US97/21475 Solutions of the active ingredients as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with surfactant, such as hydroxypropylcellulose.
Dispersions also can be prepared in glycerol, liquid polyethylene glycols, mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent growth of microorganisms. Intravenous vehicles include fluid and nutrient repienishers.
Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well-known parameters.
An effective amount of the viruses or cells is determined based on the intended goal. The term "unit dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired response in association with its administration, i. e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the subj ect to be treated, the state of the subj ect, and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.
A. Parenteral Administration The active compounds of the present invention will often be formulated for parenteral administration, e. g. , formulated for inj ection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. The preparation of an aqueous composition that contains a second agents) as active ingredients will be known to those of skill in the art in light of the present disclosure. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for inje;ctable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oiI or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
In all cases the form must be sterile and must be fluid to the extent that easy syringability exists.
It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
The active compounds may be formulated into a composition in a neutral or salt form.
Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or fernc hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the particular methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the 5 active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline 10 or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCI
solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of 15 infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
20 B. Other Routes of Administration In addition to the compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms include, e.g., tablets or other solids for oral administration; time release capsules; and any other form currently used, including cremes, lotions, mouthwashes, inhalants and the like.
The expression vectors and delivery vehicles of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route.
This includes oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
The injection can be general, regional, local or direct injection, for example, of a tumor. Also contemplated is injection of a resected tumor bed, and continuous perfusion via catheter. Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra.
The vectors of the present invention are advantageously administered in the form of injectable compositions either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior to injection also rnay be prepared. These preparations also may be emulsified. A typical compositions for such purposes comprises a 50 mg or up to about 100 mg of human serum albumin per milliliter of phosphate buffered saline. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oil and injectable organic esters, such as theyloleate.
Aqueous carriers include water, alcoholic/aqueous solutions, saline solution;>, parenteral vehicles such as sodium chloride, Ringer's dextrose, etc. Intravenous vehicle:. include fluid and nutrient replenishers.
Preservatives include antimicrobial agents, anti-oxidants, chelating agents and inert gases. The pH and exact concentration of the various components in the pharmaceutical are adjusted according to well known parameters.
Additional formulations are suitable for oral administration. Oral formulations include such typical excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. The compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders. When the route is topical, the form may be a cream, ointment, salve or spray.
An effective amount of the therapeutic agent is determined based on the intended goal.
The term "unit dose" refers to a physically discrete unit suitable for use in a subject, each unit containing a predetermined quantity of the therapeutic composition calculated to produce the desired response in association with its administration, i.e., the appropriate route and treatment regimen. The quantity to be administered, both according to number of treatments and unit dose, depends on the subject to be treated, the state of the subject and the protection desired. Precise amounts of the therapeutic composition also depend on the judgment of the practitioner and are peculiar to each individual.
incorporating the various In certain cases, the therapeutic formulations of the invention could also be prepared in forms suitable for topical administration, such as in cremes and lotions.
These forms may be used for treating skin-associated diseases, such as various sarcomas.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, with even drug release capsules and the like being employable.
C. in vitro, ex vivo) in vivo administration As used herein, the term in vitro administration refers to manipulations performed on cells removed from an animal, including, but not limited to, cells in culture.
The term ex vivo administration refers to cells which have been manipulated in vitro, and are subsequently administered to a living animal. The term in vivo administration includes all manipulations performed on cells within an animal.
In certain aspects of the present invention, the compositions may be administered either in vitro, ex vivo, or in vivo. In certain in vitro embodiments, macrophage suspension cultures are incubated with an adenoviral vector of the instant invention for 24 to 48 hours. The transduced cells can then be used for in vitro analysis, or alternatively for in vivo administration. In an additional in vitro embodiment, tumor cells are plated 24 hours prior to transduction at 10-30%
confluence. The cells are then incubated with a selected vector for 1 to 3 hours, additional medium is added, and the cells are cultured for an additional 24 to 48 hours.
The transduced cells are harvested by trypsinization, and can then be used either for analysis in vitro, or for in vivo administration.
U.S. Patents 4,690,915 and 5,199,942, both incorporated herein by reference, disclose methods for ex vivo manipulation of blood mononuclear cells and bone marrow cells for use in therapeutic applications.

WO 98/22605 PG"T/US97/21475 In vivo administration of the compositions of the present invention are also contemplated.
Examples include, but are not limited to, transduct.ion of bladder epithelium by administration of the transducing compositions of the present invention through intravesicle catheterization into the bladder (Bass et al., 1995), and transduction of liver cells by infusion of appropriate transducing compositions through the portal vein via a catheter (Bao et al., 1996). Additional examples include direct injection of tumors with the instant transducing compositions, and either intranasal or intratracheal (Dong et al., 1996) instillation of transducing compositions to effect transduction of lung cells.
D. Viruses as Therapeutic Compositions The engineered viruses of the present invention may be administered directly into animals, or alternatively administered to cells which are subsequently administered to animals.
The viruses can be combined with various of the /3-interferon inhibiting formulations to produce transducing formulations with greater transduction efficiencies.
E. Cells as Therapeutic Compositions It is proposed that engineered cells of the present invention may be introduced into animals, including human subjects, with certain needs, such as animals, including human patients, with cancer. In an exemplary, but not limiting, cancer treatment aspect, cells (preferably macrophages) are engineered to contain the gene encoding GM-CSF.
These cells are then administered to a cancer patient, home to both primary and metastatic tumors, and produce angiostatin. However, other engineered cells will also achieve advantages in accordance with the invention as described herein.
VIII. Combination Therapies In certain aspects of the present invention, the anti-cancer compositions described above can be formulated in combination with other cancer therapies, for example, but not limited to, tumor suppressor genes or other chemotherapew:ic agents. In particular embodiments of the present invention, the transduction of macrophages with GM-CSF to promote the production of angiostatin is combined with conventional cancer i:reatments such as those detailed below.

A. Tumor Suppressor Genes A large number of proteins have been reported which promote apoptosis. Among these are p53, retinoblastoma gene (Rb), Wilm's tumor (WT1), bax alpha, interleukin-lb-converting enzyme and family, MEN-1 gene (chromosome 11q13), neurofibromatosis, type 1 (NF1), cdk inhibitor p 16, colorectal cancer gene (DCC), familial adenomatosis polyposis gene (FAP), multiple tumor suppressor gene (MTS-1), BRCA1, BRCA2.
Preferred are p53 and the retinoblastoma gene. Most forms of cancer have reports of p53 mutations. Inactivation of p53 results in a failure to promote apoptosis. With this failure, cancer cells progress in tumorigenesis rather than be destined for cell death.
Providing a wild type copy of the p53 or retinoblastoma gene will promote apoptosis in cancer cells.
B. Chemotherapeutic Agents Compositions of the present invention can have an effective amount of an engineered virus or cell for therapeutic administration in combination with an effective amount of a compound (second agent) that is a chemotherapeutic agent as exemplified below.
Such compositions will generally be dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium.
A wide variety of chemotherapeutic agents may be used in combination with the therapeutic genes of the present invention. These can be, for example, agents that directly cross-link DNA, agents that intercalate into DNA, and agents that lead to chromosomal and mitotic aberrations by affecting nucleic acid synthesis.
Agents that directly cross-link nucleic acids, specifically DNA, are envisaged and are shown herein, to eventuate DNA damage leading to a synergistic antineoplastic combination.
Agents such as cisplatin, and other DNA alkylating agents may be used.
Agents that damage DNA also include compounds that interfere with DNA
replication, mitosis, and chromosomal segregation. Examples of these compounds include adriamycin (also known as doxorubicin), VP-16 (also known as etoposide), verapamil, podophyllotoxin, and the like. Widely used in clinical setting for the treatment of neoplasms these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg/m2 at 21 day intervals for adriamycin, to 35-100 mg/m2 for etoposide intravenously or orally.
1. Antibiotics 5 a. Doxorubicin Doxorubicin hydrochloride, 5,12-Naphthacenedione, (8s-cis)-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]-7,8,9,10-tetrahydro-6,8,11-trihydroxy-8-(hydroxyacetyl)-1-methoxy-hydrochloride (hydroxydaunorubicin hydrochloride, Adriamycin) is used in a wide antineoplastic spectrum. It binds to DNA and inhibits nucleic acid synthesis, inhibits mitosis and 10 promotes chromosomal aberrations.
Administered alone, it is the drug of first clhoice for the treatment of thyroid adenoma and primary hepatocellular carcinoma. It is a comp~~nent of 31 first-choice combinations for the treatment of ovarian, endometrial and breast tumors, bronchogenic oat-cell carcinoma, non-small 15 cell lung carcinoma, gastric adenocarcinoma, retinoblastoma, neuroblastoma, mycosis fungoides, pancreatic carcinoma, prostatic carcinoma, bladder carcinoma, myeloma, diffuse histiocytic lymphoma, Wilms' tumor, Hodgkin's disease, adrenal tumors, osteogenic sarcoma soft tissue sarcoma, Ewing's sarcoma, rhabdomyosarcoma and acute lymphocytic leukemia. It is an alternative drug for the treatment of islet cell, cervical, testicular and adrenocortical cancers. It is 20 also an immunosuppressant.
Doxorubicin is absorbed poorly and must be administered intravenously. The pharmacokinetics are multicompartmental. Distribution phases have half lives of 12 minutes and 3.3 hr. The elimination half life is about 30 hr. F~ orty to 50% is secreted into the bile. Most of 25 the remainder is metabolized in the liver, partly to an active metabolite (doxorubicinol), but a few percent is excreted into the urine. In the presence of liver impairment, the dose should be reduced.
Appropriate doses are, intravenous, adult, ~i0 to 75 mg/m2 at 21-day intervals or 25 to 30 30 mg/m2 on each of 2 or 3 successive days repeated at 3- or 4-wk intervals or 20 mg/m2 once a week. The lowest dose should be used in elderly patients, when there is prior bone-marrow depression caused by prior chemotherapy or neoplastic marrow invasion, or when the drug is combined with other myelopoietic suppressant drugs. The dose should be reduced by SO% if the serum bilirubin lies between 1.2 and 3 mg/dl and by 75% if above 3 mg/dl. The lifetime total dose should not exceed SSO mg/m2 in patients with normal heart function and 400 mg/m2 in persons having received mediastinal irradiation. Alternatively, 30 mg/m2 on each of 3 consecutive days, repeated every 4 wk. Exemplary doses may be 10 mg/m2, 20 mg/m2, 30 mg/m2, SO mg/m2, 100 mg/m2, 150 mg/m2, 175 mg/m2, 200 mg/m2, 225 mg/m2, 250 mg/m2, 275 mg/m2, 300 mg/m2, 350 mg/m2, 400 mg/m2, 425 mg/m2, 450 mg/m2, 475 mg/m2, mg/m2. Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
In the present invention the inventors have employed E 1 A and LT as exemplary genes for therapy to synergistically enhance the antineoplastic effects of the doxorubicin in the treatment of cancers. Those of skill in the art will be able to use the invention as exemplified potentiate the effects of doxorubicin in a range of different neu-mediated cancers.
b. Daunorubicin Daunorubicin hydrochloride, 5,12-Naphthacenedione, (8S-cis)-8-acetyl-10-[(3-amino-2,3,6-trideoxy-a-L-lyxo-hexanopyranosyl)oxy)-7,8,9,10-tetrahydro-6,8,11-trihydroxy-10 methoxy-, hydrochloride; also termed cerubidine and available from Wyeth.
Daunorubicin intercalates into DNA, blocks DAN-directed RNA polymerase and inhibits DNA
synthesis. It can prevent cell division in doses that do not interfere with nucleic acid synthesis.
In combination with other drugs it is included in the first-choice chemotherapy of acute myelocytic leukemia in adults {for induction of remission), acute lymphocytic leukemia and the acute phase of chronic myelocytic leukemia. Oral absorption is poor, and it must be given intravenously. The half life of distribution is 45 minutes and of elimination, about I9 hr. The half life of its active metabolite, daunorubicinol, is about 27 hr.
Daunorubicin is metabolized mostly in the liver and also secreted into the bile (ca 40%). Dosage must be reduced in liver or renal insuff ciencies.

Suitable doses are (base equivalent), intravenous adult, younger than 60 yr.

mg/m2/day (30 mg/m2 for patients older than 60 yr.) for 1, 2 or 3 days every 3 or 4 wk or 0.8 mg/kg/day for 3 to 6 days every 3 or 4 wk; no more than 550 mg/m2 should be given in a lifetime, except only 450 mg/m2 if there has been chest irradiation; children, 25 mg/m2 once a week unless the age is less than 2 yr. or the body surface less than 0.5 m, in which case the weight-based adult schedule is used. It is available in injectable dosage forms (base equivalent) 20 mg (as the base equivalent to 21.4 mg of the hydrochloride). Exemplary doses may be 10 mg/m2, 20 mg/m2, 30 mg/m2, 50 mg/m2, 100 mg/rn2, 150 mg/m2, 175 mg/m2, 200 mg/m2, 225 mg/m2, 250 mg/m2, 275 mg/m2, 300 mg/m2, 350 mg/m2, 400 mg/m2, 425 mg/m2, 450 mg/m2, 475 mg/m2, S00 mg/m2. Of course, all of these: dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
c. Mitomycin Mitomycin (also known as mutamycin and/or mitomycin-C) is an antibiotic isolated from 1 S the broth of Streptomyces caespitosus which has been shown to have antitumor activity. The compound is heat stable, has a high melting point, and is freely soluble in organic solvents.
Mitomycin selectively inhibits the synthesis of deoxyribonucleic acid (DNA).
The guanine and cytosine content correlates with the degree of mitomycin-induced cross-linking. At high concentrations of the drug, cellular RNA and oprotein synthesis are also suppressed.
In humans, mitomycin is rapidly cleared from the serum after intravenous administration.
Time required to reduce the serum concentration by 50% after a 30 mg. bolus injection is 17 minutes. After injection of 30 mg., 20 mg., or 10 mg. LV., the maximal serum concentrations were 2.4 mg./ml, 1.7 mg./ml, and 0.52 mg./ml, respectively. Clearance is effected primarily by metabolism in the liver, but metabolism occurs in other tissues as well. The rate of clearance is inversely proportional to the maximal serum concentration because, it is thought, of saturation of the degradative pathways.
Approximately 10% of a dose of mitomycin is excreted unchanged in the urine.
Since metabolic pathways are saturated at relatively low doses, the percent of a dose excreted in urine increases with increasing dose. In children, excretion of intravenously administered mitomycin is similar.
d. Actinomycin D
Actinomycin D (Dactinomycin) [50-76-0]; C62Hg6N12~16 (1255.43) is an antineoplastic drug that inhibits DNA-dependent RNA polymerase. It is a component of first-choice combinatipns for treatment of choriocarcinoma, embryonal rhabdomyosarcoma, testicular tumor and Wilms' tumor. Tumors which fail to respond to systemic treatment sometimes respond to local perfusion. Dactinomycin potentiates radiotherapy. It is a secondary (efferent) immunosuppressive.
Actinomycin D is used in combination with primary surgery, radiotherapy, and other drugs, particularly vincristine and cyclophosphamide. Antineoplastic activity has also been noted in Ewing's tumor, Kaposi's sarcoma, and soft-tissue sarcomas.
Dactinomycin can be effective in women with advanced cases of choriocarcinoma. It also produces consistent responses in combination with chlorambucil and in patientswithmetastatic methotrexate testicular carcinomas. A response may sometimesin patientswithHodgkin's be observed disease and non-Hodgkin's lymphomas. Dactinomycinalso usedto inhibit has been immunological responses, particularly the rej ection of renal transplants.
Half of the dose is excreted intact into the bile and 10% into the urine; the half life is about 36 hr. The drug does not pass the blood-brain barrier. Actinomycin D is supplied as a lyophilized powder (0/5 mg in each vial). The usual daily dose is 10 to 15 mg/kg; this is given intravenously for 5 days; if no manifestations of toxicity are encountered, additional courses may be given at intervals of 3 to 4 weeks. Daily injections of 100 to 400 mg have been given to children for 10 to 14 days; in other regimens, 3 to 6 mg/kg, for a total of 125 mg/kg, and weekly maintenance doses of 7.5 mg/kg have been used. Although it is safer to administer the drug into the tubing of an intravenous infusion, direct intravenous injections have been given, with the precaution of discarding the needle used to withdraw the drug from the vial in order to avoid subcutaneous reaction. Exemplary doses may be 100 mg/m2, 150 mg/m2, 175 mg/m2, mg/m2, 225 mg/m2, 250 mg/m2, 275 mg/m2, 300 mg/m2, 350 mg/m2, 400 mg/m2, 425 mg/m2, 450 mg/m2, 475 mg/m2, 500 mg/m2. Of course,, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
e. Bleomycin Bleomycin is a mixture of cytotoxic glycopeptide antibiotics isolated from a strain of Streptomyces verticillus. It is freely soluble in water.
Although the exact mechanism of action of bleomycin is unknown, available evidence would seem to indicate that the main mode of action is the inhibition of DNA
synthesis with some evidence of lesser inhibition of RNA and protein synthesis.
In mice, high concentrations of bleomycin are found in the skin, lungs, kidneys, peritoneum, and lymphatics. Tumor cells of the skin and lungs have been found to have high concentrations of bleomycin in contrast to the low concentrations found in hematopoietic tissue.
The low concentrations of bleomycin found in bone marrow may be related to high levels of bleomycin degradative enzymes found in that tissue.
In patients with a creatinine clearance o:f >35 ml per minute, the serum or plasma terminal elimination half life of bleomycin is aplnoximately 115 minutes. In patients with a creatinine clearance of <35 ml per minute, the plasma or serum terminal elimination half life increases exponentially as the creatinine clearance decreases. In humans, 60%
to 70% of an administered dose is recovered in the urine as active bleomycin.
Bleomycin should be considered a palliative treatment. It has been shown to be useful in the management of the following neoplasms either as a single agent or in proven combinations with other approved chemotherapeutic agents in squamous cell carcinoma such as head and neck (including mouth, tongue, tonsil, nasopharynx, oropharynx, sinus, palate, lip, buccal mucosa, gingiva, epiglottis, larynx), skin, penis, cervix, and vulva. It has also been used in the treatment of lymphomas and testicular carcinoma.

Because of the possibility of an anaphylactoid reaction, lymphoma patients should be treated with two units or less for the first two doses. If no acute reaction occurs, then the regular dosage schedule may be followed.
5 Improvement of Hodgkin's Disease and testicular tumors is prompt and noted within 2 weeks. If no improvement is seen by this time, improvement is unlikely.
Squamous cell cancers respond more slowly, sometimes requiring as long as 3 weeks before any improvement is noted.
Bleomycin may be given by the intramuscular, intravenous, or subcutaneous routes.
2. Miscellaneous Agents a. Cisplatin Cisplatin has been widely used to treat cancers such as metastatic testicular or ovarian carcinoma, advanced bladder cancer, head or neck cancer, cervical cancer, lung cancer or other 1 S tumors. Cisplatin can be used alone or in combination with other agents, with efficacious doses used in clinical applications of 15-20 mg/m2 for 5 days every three weeks for a total of three courses. Exemplary doses may be 0.50 mg/m2, 1.0 mg/m2, 1.50 mg/m2, 1.75 mg/m2, 2.0 mg/m2; 3.0 mg/m2 , 4.0 mg/m2, 5.0 mg/m2 , 10 mg/m2. Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
Cisplatin is not absorbed orally and must therefore be delivered via injection intravenously, subcutaneously, intratumorally or intraperitoneally.
In certain aspects of the current invention cisplatin may be used in combination with E 1 A
or LT in the treatment of breast carcinoma. It is clear, however, that the combination of cisplatin and therapeutic genes could be used for the treatment of any other neu-mediated cancer.
b. VP16 VP16 is also known as etoposide and is used primarily for treatment of testicular tumors, in combination with bleomycin and cisplatin, and in combination with cisplatin for small-cell carcinoma of the lung. It is also active against non-Hodgkin's lymphomas, acute nonlymphocytic leukemia, carcinoma of the breast, and Kaposi's sarcoma associated with acquired immunodeficiency syndrome (AIDS).
VP 16 is available as a solution (20 mg/ml) for intravenous administration and as SO-mg, liquid-filled capsules for oral use. For small-cell carcinoma of the lung, the intravenous dose (in combination therapy) is can be as much as 100 mg/m2 or as little as 2 mg/ m2, routinely 35 mg/m2, daily for 4 days, to 50 mg/m2, daily for 5 days have also been used.
When given orally, the dose should be doubled. Hence the doses for small cell lung carcinoma may be as high as 200-250 mg/m2. The intravenous dose for testicular cancer (in combination therapy) is 50 to 100 mg/m2 daily for 5 days, or 100 mg/m2 on alternate days, for three doses.
Cycles of therapy are usually repeated every 3 to 4 weeks. The drug, should be administered slowly during a 30- to 60-minute infusion in order to avoid hypotension and bronchospasm, which are probably due to the solvents used in the formulation.
1 S c. Tumor Necrosis Factor Tumor Necrosis Factor [TNF; Cachectin) is a glycoprotein that kills some kinds of cancer cells, activates cytokine production, activates macrophages and endothelial cells, promotes the production of collagen and collagenases, is an inflammatory mediator and also a mediator of septic shock, and promotes catabolism, fever an~i sleep. Some infectious agents cause tumor regression through the stimulation of TNF production. TNF can be quite toxic when used alone in effective doses, so that the optimal regimens probably will use it in lower doses in combination with other drugs. Its immunosup~pressive actions are potentiated by gamma-interferon, so that the combination potentially is dangerous. A hybrid of TNF
and interferon-a also has been found to possess anti-cancer activity, 3. Plant Alkaloids a. Taxol Taxol is an experimental antimitotic agent, isolated from the bark of the ash tree, Taxus brevifolia. It binds to tubulin (at a site distincl: from that used by the vinca alkaloids) and promotes the assembly of microtubules. Taxol is currently being evaluated clinically; it has activity against malignant melanoma and carcinoma of the ovary. Maximal doses are 30 mg/m2 per day for S days or 210 to 250 mg/m2 given once every 3 weeks. Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
b. Vincristine Vincristine blocks mitosis and produces metaphase arrest. It seems likely that most of the biological activities of this drug can be explained by its ability to bind specifically to tubulin and to block the ability of protein to polymerize into microtubules. Through disruption of the microtubules of the mitotic apparatus, cell division is arrested in metaphase.
The inability to segregate chromosomes correctly during mitosis presumably leads to cell death.
The relatively low toxicity of vincristine for normal marrow cells and epithelial cells make this agent unusual among anti-neoplastic drugs, and it is often included in combination with other myelosuppressive agents.
Unpredictable absorption has been reported after oral administration of vinblastine or vincristine. At the usual clinical doses the peak concentration of each drug in plasma is approximately 0.4 mM.
Vinblastine and vincristine bind to plasma proteins. They are extensively concentrated in platelets and to a lesser extent in leukocytes and erythrocytes.
Vincristine has a multiphasic pattern of clearance from the plasma; the terminal half life is about 24 hours. The drug is metabolized in the liver, but no biologically active derivatives have been identified. Doses should be reduced in patients with hepatic dysfunction. At least a 50% reduction in dosage is indicated if the concentration of bilirubin in plasma is greater than 3 mg/dl (about 50 mM).
Vincristine sulfate is available as a solution (1 mg/ml) for intravenous injection.
Vincristine used together with corticosteroids is presently the treatment of choice to induce remissions in childhood leukemia; the optimal dosages for these drugs appear to be vincristine, intravenously, 2 mg/m2 of body-surface area, weekly, and prednisone, orally, 40 mg/m2, daily.

Adult patients with Hodgkin's disease or non-Hodgkin's lymphomas usually receive vincristine as a part of a complex protocol. When used in t:he MOPP regimen, the recommended dose of vincristine is 1.4 mg/m2. High doses of vincristine seem to be tolerated better by children with leukemia than by adults, who may experience sever neurological toxicity.
Administration of the S drug more frequently than every 7 days or apt higher doses seems to increase the toxic manifestations without proportional improvement in the response rate.
Precautions should also be used to avoid extravasation during intravenous administration of vincristine. Vincristine (and vinblastine) can be infused into the arterial blood supply of tumors in doses several times larger than those that can be administered intravenously with comparable toxicity.
Vincristine has been effective in Hodgkiri's disease and other lymphomas.
Although it appears to be somewhat less beneficial than vinbl.astine when used alone in Hodgkin's disease, when used with mechlorethamine, prednisone, and procarbazine (the so-called MOPP regimen), it is the preferred treatment for the advanced stage; (III and IV) of this disease. In non-Hodgkin's lymphomas, vincristine is an important agent, particularly when used with cyclophosphamide, bleomycin, doxorubicin, and prednisone. Vin~~ristine is more useful than vinblastine in lymphocytic leukemia. Beneficial response have >=peen reported in patients with a variety of other neoplasms, particularly Wilms' tumor, neuroblastoma, brain tumors, rhabdomyosarcoma, and carcinomas of the breast, bladder, and the male and female reproductive systems.
Doses of vincristine for use will be deaermined by the clinician according to the individual patients need. 0.01 to 0.03 mg/kg or 0.4~ to 1.4 mg/m2 can be administered or 1.5 to 2 mg/m2 can also be administered. Alternatively 0.02 mg/m2, 0.05 mg/m2, 0.06 mg/m2, 0.07 mg/m2, 0.08 mg/m2, 0.1 mg/m2, O.I2 mg/m2, 0.14 mg/m2, 0.15 mg/m2, 0.2 mg/m2, 0.25 mg/m2 can be given as a constant intravenous infusion. Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention.
c. Vinblastine When cells are incubated with vinblastine, dissolution of the microtubules occurs.
Unpredictable absorption has been reported after oral administration of vinblastine or vincristine.
At the usual clinical doses the peak concentration of each drug in plasma is approximately 0.4 mM. Vinblastine and vincristine bind to plasma proteins. They are extensively concentrated in platelets and to a lesser extent in leukocytes and erythrocytes.
After intravenous injection, vinblastine has a multiphasic pattern of clearance from the plasma; after distribution, drug disappears from plasma with half lives of approximately 1 and 20 hours.
Vinblastine is metabolized in the liver to biologically activate derivative desacetylvinblastine. Approximately 15% of an administered dose is detected intact in the urine, and about 10% is recovered in the feces after biliary excretion. Doses should be reduced in patients with hepatic dysfunction. At least a 50% reduction in dosage is indicated if the concentration of bilirubin in plasma is greater than 3 mg/dl (about SO mM).
Vinblastine sulfate is available in preparations for injection. The drug is given intravenously; special precautions must be taken against subcutaneous extravasation, since this may cause painful irritation and ulceration. The drug should not be injected into an extremity with impaired circulation. After a single dose of 0.3 mg/kg of body weight, myelosuppression reaches its maximum in 7 to 10 days. If a moderate level of leukopenia (approximately 3000 cells/mm3) is not attained, the weekly dose may be increased gradually by increments of 0.05 mg/kg of body weight. In regimens designed to cure testicular cancer, vinblastine is used in doses of 0.3 mg/kg every 3 weeks irrespective of blood cell counts or toxicity.
The most important clinical use of vinblastine is with bleomycin and cisplatin in the curative therapy of metastatic testicular tumors. Beneficial responses have been reported in various lymphomas, particularly Hodgkin's disease, where significant improvement may be noted in 50 to 90% of cases. The effectiveness of vinblastine in a high proportion of lymphomas is not diminished when the disease is refractory to alkylating agents. It is also active in Kaposi's sarcoma, neurobiastoma, and Letterer-Siwe disease (histiocytosis X), as well as in carcinoma of the breast and choriocarcinoma in women.
Doses of vinblastine for use will be determined by the clinician according to the individual patients need. 0.1 to 0.3 mg/kg can be administered or 1.5 to 2 mg/m2 can also be administered. Alternatively, 0.1 mg/m2, 0.12 mg/m2, 0.14 mg/m2, 0.15 mg/m2, 0.2 mg/m2, 0.25 mg/m2, 0.5 mg/m2, 1.0 mg/m2, 1.2 mg/m2, 1.4 mg/m2, 1.5 mg/m2, 2.0 mg/m2, 2.5 mg/m2, 5.0 mg/m2, 6 mg/m2, 8 mg/m2, 9 mg/m2, 10 mg/m~Z, 20 mg/m2, can be given. Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of 5 use in the invention.
4. Allrylating Agents a. Carmustine Carmustine (sterile carmustine) is one of the nitrosoureas used in the treatment of certain 10 neoplastic diseases. It is 1,3 bis (2-chloroethyl;l-1-nitrosourea. It is lyophilized pale yellow flakes or congealed mass with a molecular weighl: of 214.06. It is highly soluble in alcohol and lipids, and poorly soluble in water. Carmustine is administered by intravenous infusion after reconstitution as recommended. Sterile carmustin~~ is commonly available in 100 mg single dose vials of lyophilized material.
Although it is generally agreed that carmustine alkylates DNA and RNA, it is not cross resistant with other alkylators. As with other nitrosoureas, it may also inhibit several key enzymatic processes by carbamoylation of amino acids in proteins.
Carmustine is indicated as palliative therapy as a single agent or in established combination therapy with other approved chemotherapeutic agents in brain tumors such as glioblastoma, brainstem glioma, medullobladyoma, astrocytoma, ependymoma, and metastatic brain tumors. Also it has been used in combination with prednisone to treat multiple myeloma.
Carmustine has proved useful, in the treatment of Hodgkin's Disease and in non-Hodgkin's lymphomas, as secondary therapy in combinatio n with other approved drugs in patients who relapse while being treated with primary therapy, or who fail to respond to primary therapy.
The recommended dose of carmustine as a single agent in previously untreated patients is 150 to 200 mg/m2 intravenously every 6 weeks. This may be given as a single dose or divided into daily injections such as 75 to 100 mg/m2 on l successive days. When carmustine is used in combination with other myelosuppressive drugs or in patients in whom bone marrow reserve is depleted, the doses should be adjusted accordingly. Doses subsequent to the initial dose should be adjusted according to the hematologic response of the patient to the preceding dose. It is of course understood that other doses may be used in the present invention for example 10 mg/m2, 20 mg/m2, 30 mg/m2, 40 mg/m2, 50 mg/m2, 60 mg/m2, 70 mg/m2, 80 mg/m2, 90 mg/m2, or 100 mg/m2 . The skilled artisan is directed to, "Remington's Pharmaceutical Sciences" ISth Edition, chapter 61. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject:
b. MelphaIan Melphalan also known as alkeran, L-phenylalanine mustard, phenylalanine mustard, L-PAM, or L-sarcolysin, is a phenylalanine derivative of nitrogen mustard.
Melphalan is a bifunctional alkylating agent which is active against selective human neoplastic diseases. It is known chemically as 4-jbis(2-chloroethyl)amino]-L-phenylalanine.
Melphalan is the active L-isomer of the compound and was first synthesized in 1953 by Bergel and Stock; the D-isomer, known as medphalan, is less active against certain animal tumors, and the dose needed to produce effects on chromosomes is larger than that required with the L-isomer. The racemic (DL-) form is known as merphalan or sarcolysin.
Melphalan is insoluble in water and has a pKa 1 of ~2.1. Melphalan is available in tablet form for oral administration and has been used to treat multiple myeloma.
Available evidence suggests that about one third to one half of the patients with multiple myeloma show a favorable response to oral administration of the drug.
Melphalan has been used in the treatment of epithelial ovarian carcinoma. One commonly employed regimen for the treatment of ovarian carcinoma has been to administer melphalan at a dose of 0.2 mg/kg daily for five days as a single course.
Courses are repeated every four to five weeks depending upon hematologic tolerance (Smith and Rutledge, 1975;
Young et al., 1978). Alternatively the dose of melphalan used could be as low as 0.05 mg/kg/day or as high as 3 mg/kg/day or any dose in between these doses or above these doses. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
c. Cyclophosphamide Cyclophosphamide is 2H 1,3,2-Oxazapl:losphorin-2-amine, N,N bis (2-chloroethyl) tetrahydro-, 2-oxide, monohydrate; termed Cytox;an available from Mead Johnson; and Neosar available from Adria. Cyclophosphamide is pre~~ared by condensing 3-amino-1-propanol with N,N-bis(2-chlorethyl) phosphoramidic dichloride ~:(C1CH2CH2)2N--POC12] in dioxane solution under the catalytic influence of triethylamine. The condensation is double, involving both the hydroxyl and the amino groups, thus effecting the cyclization.
Unlike other (3-chloroethylamino alkylators, it does not cyclize readily to the active ethyleneimonium form until activated by hepatic enzymes. Thus, the substance is stable in the gastrointestinal tract, tolerated well and effective by the oral and parental routes and does not cause local vesication, necrosis, phlebitis or even pain.
Suitable doses for adults include, orally, 1 to 5 mg/kg/day (usually in combination), depending upon gastrointestinal tolerance; or 1 t~~ 2 mg/kg/day;
intravenously, initially 40 to 50 mg/kg in divided doses over a period of 2 to S clays or 10 to 15 mg/kg every 7 to 10 days or 3 to 5 mg/kg twice a week or 1.5 to 3 mg/kg/day . E, dose 250 mg/kg/day may be administered as an antineoplastic. Because of gastrointestinal adverse effects, the intravenous route is preferred for loading. During maintenance, a leukocyte count of 3000 to 4000/mm3 usually is desired.
The drug also sometimes is administered intramuscularly, by infiltration or into body cavities. It is available in dosage forms for injection of 100, 200 and 500 mg, and tablets of 25 and 50 mg the skilled artisan is referred to "Remington's Pharmaceutical Sciences" 15th Edition, chapter 61, incorporate herein as a reference, for details on doses for administration.
d. Chlorambucil Chlorambucil (also known as leukeran) wa,s first synthesized by Everett et al.
( 1953). It is a bifunctional alkylating agent of the nitrogen mustard type that has been found active against selected human neoplastic diseases. Chlorambucil is known chemically as 4-[bis(2-chlorethyl)amino) benzenebutanoic acid.
Chlorambucil is available in tablet form for oral administration. It is rapidly and completely absorbed from the gastrointestinal tract. After single oral doses of 0.6-1.2 mg/kg, peak plasma chlorambucil levels are reached within one hour and the terminal half life of the parent drug is estimated at 1.5 hours. 0.1 to 0.2 mg/kg/day or 3 to 6 mg/m2/day or alternatively 0.4 mg/kg may be used for antineoplastic treatment. Treatment regimes are well know to those of skill in the art and can be found in the "Physicians Desk Reference" and in "Remingtons Pharmaceutical Sciences" referenced herein.
Chlorambucil is indicated in the treatment of chronic lymphatic (lymphocytic) leukemia, malignant lymphomas including lymphosarcoma, giant follicular lymphoma and Hodgkin's disease. It is not curative in any of these disorders but may produce clinically useful palliation.
e. Busulfan Busulfan (also known as myleran) is a bifunctional alkyiating agent. Busulfan is known chemically as 1,4-butanediol dimethanesulfonate.
Busulfan is not a structural analog of the nitrogen mustards. Busulfan is available in tablet form for oral administration. Each scored tablet contains 2 mg busulfan and the inactive ingredients magnesium stearate and sodium chloride.
Busulfan is indicated for the palliative treatment of chronic myelogenous (myeloid, myelocytic, granulocytic) leukemia. Although not curative, busulfan reduces the total granulocyte mass, relieves symptoms of the disease, and improves the clinical state of the patient. Approximately 90% of adults with previously untreated chronic myelogenous leukemia will obtain hematologic remission with regression or stabilization of organomegaly following the use of busulfan. It has been shown to be superior to splenic irradiation with respect to survival times and maintenance of hemoglobin levels, and to be equivalent to irradiation at controlling splenomegaly.

f. Lomustine Lomustine is one of the nitrosoureas used in the treatment of certain neoplastic diseases.
It is 1-(2-chloro-ethyl)-3-cyclohexyl-1 nitrosourea. It is a yellow powder with the empirical formula of C9H16C1N302 and a molecular weight of 233.71. Lomustine is soluble in 10%
ethanol (0.05 mg per ml) and in absolute alcohol (70 mg per ml). Lomustine is relatively insoluble in water (<0.05 mg per ml). It is relatively unionized at a physiological pH. Inactive ingredients in lomustine capsules are: magnesium stearate and mannitol.
Although it is generally agreed that lomustine alkylates DNA and RNA, it is not cross resistant with other alkylators. As with other nitrosoureas, it may also inhibit several key enzymatic processes by carbamoylation of amino acids in proteins.
Lomustine may be given orally. Following oral administration of radioactive lomustine at doses ranging from 30 mg/m2 to 100 mg/m2, about half of the radioactivity given was excreted in the form of degradation products within 24 hours.
The serum half life of the metabolites ranl;es from 16 hours to 2 days. Tissue levels are comparable to plasma levels at 15 minutes after intravenous administration.
Lomustine has been shown to be useful as a single agent in addition to other treatment modalities, or in established combination therapy with other approved chemotherapeutic agents in both primary and metastatic brain tumors, in patients who have already received appropriate surgical and/or radiotherapeutic procedures. It has also proved effective in secondary therapy against Hodgkin's Disease in combination with other approved drugs in patients who relapse while being treated with primary therapy, or who fail to respond to primary therapy.
The recommended dose of lomustine in adults and children as a single agent in previously untreated patients is 130 mg/m2 as a single oral dose every 6 weeks. In individuals with compromised bone marrow function, the dose should be reduced to 100 mg/m2 every 6 weeks. When lomustine is used in combination with other myelosuppressive drugs, the doses should be adjusted accordingly. It is understood that other doses may be used for example, 20 WO 98!22605 PCT/LTS97/21475 mg/m2 30 mg/m2, 40 mg/m2, 50 mg/m2, 60 mg/m2, 70 mg/m2, 80 mg/m2, 90 mg/m2, mg/m2, 120 mg/m2 or any doses between these ranges as determined by the clinician to be necessary for the individual being treated.
5 IX Use of Transduced Cells in Bioreactors The abili"y to produce biologically active polypeptides is increasingly important to the pharmaceutical industry. The present invention discloses compositions and methods for the efficient transduction of cells, allowing for the production of proteins in vitro from previously 10 refractory cell types.
Over the last decade, advances in biotechnology have led to the production of important proteins and factors from bacteria, yeast, insect cells and from mammalian cell culture.
Mammalian cultures have advantages over cultures derived from the less advanced lifeforms in 15 their ability to post-translationally process complex protein structures such as disulfide-dependent folding and glycosylation. Indeed, mammalian cell culture is now the preferred source of a number of important proteins for use in human and animal medicine, especially those which are relatively large, complex or glycosylated.
20 Development of mammalian cell culture for production of pharmaceuticals has been greatly aided by the development in molecular biology of techniques for design and construction of vector systems highly efficient in mammalian cell cultures, a battery of useful selection markers, gene amplification schemes and a more comprehensive understanding of the biochemical and cellular mechanisms involved in procuring the final biologically-active 25 molecule from the introduced vector.
However, the traditional selection of cell types for expressing heterologous proteins has generally been limited to the more "common" cell types such as CHO cells, BHK
cells, C 127 cells and myeloma cells. In many cases, these cell types were selected because there was a great 30 deal of preexisting literature on the cell type or the cell was simply being carried in the laboratory at the time the effort was made to express a peptide product.
Frequently, factors which affect the downstream (e.g., beyond the T-75 flask) side of manufacturing scale-up were not considered before selecting the cell line as the :host for the expression system.
Aspects of the present invention take advantage of the biochemical and cellular capacities of mammalian cells as well as of recently available bioreactor technology.
Growing cells according to the present invention in a bioreactor ~~llows for large scale production and secretion of complex, fully biologically-active polypepti~des into the growth media. In particular embodiments, by designing a defined media with low contents of complex proteins and using a scheme of timed-stimulation of the secretion into the media for increased titer, the purification strategy can be greatly simplified, thus lowering production cost.
1. Anchorage-dependent ver~;us non-anchorage-dependent cultures.
Animal and human cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing freely in suspension throughout the bulk of the culture; or as anchorage dependent cells requiring attachment to a solid suhstrate for their propagation (i. e., a monolayer type of cell growth).
Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products. Large scale suspension culture based on microbial (bacterial and yeast} fermentation technology has clear advantages for the manufacturing of mammalian cell products. The processes are relatively straightforward to operate and scale up. Homogeneous conditions can be provided in the reactor which allows for precise monitoring and control of temperature, dissolved oxygen, and pH, and ensure that representative samples of the culture can be taken.
However, suspension cultured cells cannot always be used in the production of biologicals. Suspension cultures are still considered to have tumorigenic potential and thus their use as substrates for production put limits on the use of the resulting products in human and veterinary applications (Petricciani, 1985; Larsson and Litwin, 1987). Viruses propagated in suspension cultures as opposed to anchorage-dependent cultures can sometimes cause rapid changes in viral markers, leading to reduced immunogenicity (Bahnemann, 1980).
Finally, sometimes even recombinant cell lines can secrete considerably higher amounts of products WO 98/22605 PCT/iJS97/21475 when propagated as anchorage-dependent cultures as compared with the same cell line in suspension (Nilsson and Mosbach, 1987). For these reasons, different types of anchorage-dependent cells are used extensively in the production of different biological products.
The current invention includes cells which are anchorage-dependent of nature.
Anchorage-dependent cells, when grown in suspension, will attach to each other and grow in clumps, eventually suffocating cells in the inner core of each clump as they reach a size that leaves the core cells unsustainable by the culture conditions. Therefore, an efficient means of large-scale culture of anchorage-dependent cells is also provided in order to effectively take advantage of the cells' capacity to secrete heterologous proteins.
2. Reactors and processes for suspension.
Large scale suspension culture of mammalian cultures in stirred tanks is contemplated.
The instrumentation and controls for bioreactors have been adapted, along with the design of the fermentors, from related microbial applications. However, acknowledging the increased demand for contamination control in the slower growing mammalian cultures, improved aseptic designs have been implemented, improving dependability of these reactors.
Instrumentation and controls include agitation, temperature, dissolved oxygen, and pH controls. More advanced probes and autoanalyzers for on-line and off line measurements of turbidity (a function of particles present), capacitance (a function of viable cells present), glucose/lactate, carbonate/bicarbonate and carbon dioxide are also available. Maximum cell densities obtainable in suspension cultures are relatively low at about 2-4 ' 106 cells/ml of medium (which is less than 1 mg dry cell weight per ml), well below the numbers achieved in microbial fermentation.
Two suspension culture reactor designs are most widely used in the industry due to their simplicity and robustness of operation - the stirred reactor and the airlift reactor. The stirred reactor design has successfully been used on a scale of 8000 liter capacity for the production of interferon (Phillips et al., 1985; Mizrahi, 1983). Cells are grown in a stainless steel tank with a height-to-diameter ratio of 1:1 to 3:1. The culture is usually mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stirrer shafts.
The airlift reactor, also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture. The gas stream enters a riser section of the reactor and drives circulation. Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor. The main advantage of this dc;sign is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10:1. The airlift reactor scales up relatively readily, has good mass transfer of gasses and generates relatively low shear forces.
Most large-scale suspension cultures are operated as batch or fed-batch processes because they are the most straightforward to operate and scale up. However, continuous processes based on chemostat or perfusion principles are available.
A batch process is a closed system in which a typical growth profile is seen.
A lag phase is followed by exponential, stationary and decline phases. In such a system, the environment is continuously changing as nutrients are deplete~3 and metabolites accumulate.
This makes analysis of factors influencing cell growth and productivity, and hence optimization of the process, a complex task. Productivity of a batch Inocess may be increased by controlled feeding of key nutrients to prolong the growth cycle. Such a fed-batch process is still a closed system because cells, products and waste products are not removed.
In what is still a closed system, perfusion of fresh medium through the culture can be achieved by retaining the cells with a fine mesh spin filter and spinning to prevent clogging.
Spin filter cultures can produce cell densities of approximately 5 x 10~
cells/ml. A true open system and the most basic perfusion process is the chemostat in which there is an inflow of medium and an outflow of cells and products. Culture medium is fed to the reactor at a predetermined and constant rate which maintains the dilution rate of the culture at a value less than the maximum specific growth rate of the cells (to prevent washout of the cell mass from the reactor). Culture fluid containing cells, cell products and byproducts is removed at the same rate.
These perfused systems are not in commercial use for production from mammalian cell culture.

3. Non-perfused attachment systems.
Traditionally, anchorage-dependent cell cultures are propagated on the bottom of small glass or plastic vessels. The restricted surface-to-volume ratio offered by classical and traditional techniques, suitable for the laboratory scale, has created a bottleneck in the production of cells and cell products on a large scale. To provide systems that offer large accessible surfaces for cell growth in small culture volume, a number of techniques have been proposed: the roller bottle system, the stack plates propagator, the spiral film bottles, the hollow fiber system, the packed bed, the plate exchanger system, and the membrane tubing reel. Since these systems are non-homogeneous in their nature, and are sometimes based on multiple processes, they can sometimes have limited potential for scale-up, difficulties in taking cell samples, limited potential for measuring and controlling the system and difficulty in maintaining homogeneous environmental conditions throughout the culture.
1 S A commonly used process of these systems is the roller bottle. Being little more than a large, differently shaped T-flask, simplicity of the system makes it very dependable and, hence, attractive. Fully automated robots are available that can handle thousands of roller bottles per day, thus eliminating the risk of contamination and inconsistency associated with the otherwise required intense human handling. With frequent media changes, roller bottle cultures can achieve cell densities of close to 0.5 x 106 cells/cm2 (corresponding to 109 cells/bottle or 107 cells/ml of culture media).
4. Cultures on microcarriers van Wezel (1967) developed the concept of the microcarrier culturing systems.
In this system, cells are propagated on the surface of small solid particles suspended in the growth medium by slow agitation. Cells attach to the microcarriers and grow gradually to confluency of the microcarrier surface. In fact, this large scale culture system upgrades the attachment dependent culture from a single disc process to a unit process in which both monolayer and suspension culture have been brought together. Thus, combining the necessary surface for the cells to grow with the advantages of the homogeneous suspension culture increases production.

The advantages of microcarrier cultures over most other anchorage-dependent, large-scale cultivation methods are several fold. First, microcarrier cultures offer a high surface-to-volume ratio (variable by changing the Garner concentration) which leads to high cell density yields and a potential for obtaining highly concentrated cell products. Cell yields are up to 1-2 x 5 10~ cells/ml when cultures are propagated in a ~perfused reactor mode.
Second, cells can be propagated in one unit process vessels instead of using many small low-productivity vessels (i. e.) flasks or dishes). This results in far better utilization and a considerable saving of culture medium. Moreover, propagation in a single reactor leads to reduction in need for facility space and in the number of handling steps required per cell, thus reducing labor cost and risk of 10 contamination.
Third, the well-mixed and homogeneous microcarrier suspension culture makes it possible to monitor and control environmental conditions (e.g., pH, p02, and concentration of medium components), thus leading to more reproducible cell propagation and product recovery.
1 S Fourth, it is possible to take a representative s~unple for microscopic observation, chemical testing, or enumeration. Fifth, since microcarriers settle out of suspension easily, use of a fed-batch process or harvesting of cells can be done relatively easily. Sixth, the mode of the anchorage-dependent culture propagation on the microcarners makes it possible to use this system for other cellular manipulations, such as cell transfer without the use of proteolytic 20 enzymes, cocultivation of cells, transplantation unto animals, and perfusion of the culture using decanters, columns, fluidized beds, or hollow fibers for microcarrier retainment. Seventh, microcarner cultures are relatively easily scaled up using conventional equipment used for cultivation of microbial and animal cells in suspension.
25 5. Microencapsulation of mammalian cells One method which has shown to be particularly useful for culturing mammalian cells is microencapsulation. The mammalian cells are retained inside a semipermeable hydrogel membrane. A porous membrane is formed around the cells permitting the exchange of nutrients, gases, and metabolic products with the bulk medium surrounding the capsule.
Several methods 30 have been developed that are gentle, rapid and non-toxic and where the resulting membrane is sufficiently porous and strong to sustain the growing cell mass throughout the term of the culture. These methods are all based on soluble alginate gelled by droplet contact with a calcium-containing solution. Lim ( 1982) describes cells concentrated in an approximately 1 solution of sodium alginate which are forced through a small orifice, forming droplets, and breaking free into an approximately 1 % calcium chloride solution. The droplets are then cast in a layer of polyamino acid that ionically bonds to the surface alginate.
Finally the alginate is reliquefied by treating the droplet in a chelating agent to remove the calcium ions. Other methods use cells in a calcium solution to be dropped into a alginate solution, thus creating a hollow alginate sphere. A similar approach involves cells in a chitosan solution dropped into alginate, also creating hollow spheres.
Microencapsulated cells are easily propagated in stirred tank reactors and, with beads sizes in the range of 150-1500 mm in diameter, are easily retained in a perfused reactor using a fine-meshed screen. The ratio of capsule volume to total media volume can kept from as dense as 1:2 to 1:10. With intracapsular cell densities of up to 108, the effective cell density in the culture is 1-S x 10~.
The advantages of microencapsulation over other processes include the protection from the deleterious effects of shear stresses which occur from sparging and agitation, the ability to easily retain beads for the purpose of using perfused systems, scale up is relatively straightforward and the ability to use the beads for implantation.
6. Perfused attachment systems Perfusion refers to continuous flow at a steady rate, through or over a population of cells (of a physiological nutrient solution). It implies the retention of the cells within the culture unit as opposed to continuous-flow culture which washes the cells out with the withdrawn media (e.g.) chemostat). The idea of perfusion has been known since the beginning of the century, and has been applied to keep small pieces of tissue viable for extended microscopic observation. The technique was initiated to mimic the cells milieu in vivo where cells are continuously supplied with blood, lymph, or other body fluids. Without perfusion, cells in culture go through alternating phases of being fed and starved, thus limiting full expression of their growth and metabolic potential. The current use of perfused culture is to grow cells at high densities (i. e., 0.1-5 x 108 cells/ml). In order to increase densities beyond 2-4 ' 106 cells/ml (or 2 x 1 OS

cells/cm2), the medium has to be constantly replaced with a fresh supply in order to make up for nutritional deficiencies and to remove toxic products. Perfusion allows for a far better control of the culture environment (pH, p02, nutrient levels, ntc. ) and is a means of significantly increasing the utilization of the surface area within a culture for cell attachment.
Microcarrier and microencapsulated cultures are readily adapted to perfused reactors but, as noted above, these culture methods lack the capacity to meet the demand for cell densities above 10g cells/ml. Such densities will provide ;for the advantage of high product titer in the medium (facilitating downstream processing), a smaller culture system (lowering facility needs), and a better medium utilization (yielding savings in serum and other expensive additives).
Supporting cells at high density requires efficient perfusion techniques to prevent the development of non-homogeneity.
The cells of the present invention may, irre:~pective of the culture method chosen, be used in protein production and as cells for in vitro ~;ellular assays and screens as part of drug development protocols.
7. Protein Purification Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide.
The term "purified protein or peptide " as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state. A purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
Generally, "purified" will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activil:y. Where the term "substantially purified" is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50% or more of the proteins in the composition.

Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure.
These include, for example, determining the specific activity of an active fraction, or assessing the number of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number". The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation;
chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography;
isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater -fold purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.
High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain and adequ~~te flow rate. Separation can be accomplished in a matter of minutes, or a most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and clo~;e-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, I S separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc.
There also is virtually no adsorption, less zone spreading and the elution volume is related in a simple matter to molecular weight.
Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance fiom the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc. ).
A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that WO 98/22b05 PCT/US97/21475 bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavaiin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin.
Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography.
X. Kits All the essential materials and reagents required for the various aspects of the present invention may be assembled together in a kit. When the components of the kit are provided in one or more liquid solutions, the liquid solution preferably is an aqueous solution, with a sterile aqueous solution being particularly preferred.
For in vivo use, the instant compositions may be formulated into a single or separate pharmaceutically acceptable syringeable composition. In this case, the container means may itself be an inhalant, syringe, pipette, eye dropper, or other such like apparatus, from which the formulation may be applied to an infected area of the body, such as the lungs, injected into an animal, or even applied to and mixed with the other components of the kit.

The components of the kit may also be provided in dried or lyophilized forms.
When reagents or components are provided as a dried farm, reconstitution generally is by the addition of a suitable solvent. It is envisioned that the solvent also may be provided in another container means. The kits of the invention may also include an instruction sheet defining administration of the gene therapy and/or the chemotherapeutic drug.
The kits of the present invention also will typically include a means for containing the vials in close confinement for commercial sale such as, e.g., injection or blow-molded plastic containers into which the desired vials are retained. Irrespective of the number or type of containers, the kits of the invention also may corr~prise, or be packaged with, an instrument for assisting with the injection/administration or placement of the ultimate complex composition within the body of an animal. Such an instrument: may be an inhalant, syringe, pipette, forceps, measured spoon, eye dropper or any such medically approved delivery vehicle.
Additionally, instructions for use of the kit components is typical'.ly included.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art shouldl, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Adenoviral vectors induce expression of IFN-Q
Specific pathogen-free female C57BL/6 mice were purchased from Jackson Laboratory, Bar Harbor, Maine. The mice were used when 8-12 weeks of age according to institutional guidelines. Animals were maintained in facilitie~~ approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the United States Department of Agriculture, Department of Health and Human Services, and National Institutes of Health.

Macrophages were collected by peritoneal lavage from mice given an i.p.
injection of 1.5 ml of thioglycollate broth (Baltimore Biological Laboratories, Cockeysville, Maryland) 4 days before harvest (Zhang et al., 1994a). The macrophages were washed with Ca2+-and Mg2+-free phosphate buffered saline and resuspended in serum-free minimum essential medium (MEM), and 1 x 1 OS cells in 0.2 ml MEM were plated into 38-mm2 wells of 96-well flat-bottomed Microtest III
plates (Falcon Plastics, Oxford, California). After 90 min, the wells were washed with HBSS to remove nonadherent cells. The resultant macrophage monolayer was >98% pure according to morphologic and phagocytic criteria.
The plasmids pxCMV and pJMl7, AdSCMV-LacZ (a recombinant adenovirus encoding the E. toll LacZ gene) and the human transformed primary embryonic kidney 293 cell line were obtained from Dr. W. Zhang (M. D. Anderson Cancer Center) (Chu et al., 1992).
The full coding region of human IFN-(3 cDNA was subcloned into a pxCMV plasmid to generate the shuttle vector pEC-HuIFN-(3. AdSCMV-HuIFN-~i was obtained by homologous recombination of pEC-HuIFN-~3 and plasmid pJMl7 in line 293 cells using liposome-mediated transfection with lipofectin. The vectors were propagated in 293 cells. The viruses released from the infected 293 cells by 3 cycles of freezing-thawing were used without further purification to avoid endotoxin contamination.
However, similar results were also obtained using the vectors purified by double CsCI banding.
The virus titers were measured in 293 cells using a plaque assay (Graham and Prevec, 1991 ).
Mouse peritoneal macrophages (bong et al., 1993a) or NIH 3T3 cells were incubated with AdSCMV-LacZ, a recombinant adenovirus carrying an Escherichia toll (3-galactosidase (~3-gal) gene under the control of a cytomegalovirus promoter (Zhang et al., 1994b). Total cellular RNA was extracted and expression of mIFN-~3 was analyzed in control and virus-infected macrophages by reverse transcriptase-polymerase chain reaction (RT-PCRTM).
Primers for (3-actin were added into the same reaction vial to evaluate sample loading.
Total cellular RNA was extracted from control or infected cells. The RNA was treated for 30 min at 37°C with 1 U/mg RQ 1 RNase-free DNase (Promega, Madison, Wisconsin), and 1 mg of the treated RNA was reverse-transcribed for 1 S min at 42°C using an AMV reverse transcriptase system (Promega) in a final volume of 20 ml. Resulting cDNA (5 ml/reaction) was amplified with 2.5 U of Taq polymerase in a 50 ml reaction volume containing 10 mM Tris-HC1, pH 9.0 at 25°C, 1.5 mM MgCl2, 50 mM KCI, 0.1 % Triton X-100, 200 mM concentrations of each of the dNTPs, and 200 nM each of primers 1 to 4.
Primer 1 (CCAAGAAAGGACGAACATT; SEQ ID NO:1) and primer 2 (ATCTCTGCTCGGACCACCA; SEQ ID N0:2) define a 411-by fragment of mIFN-Vii.
Primer 3 (GTGGGCCGCTCTAGGCACCA; SEQ ID N0:3) and primer 4 (CGGTTGGCCTTAGGGGTCAGGCTGG;SEQ ID N0:4) define a 245-by fragment of mouse ~i-actin (Stratagene, La Jolla, California). Amplification was carried out on a Perkin-Elmer-Cetus thermal cycler for 25 cycles { 1 cycle = 94°C, 45 s; 60°C, 45 s;
and 72°C, 1 min). Thirty-microliter aliquots of each resulting mixture were separated on 1.5% agarose gel and viewed under ultraviolet (UV) light (for (3-actin). The separated DNA was then transferred onto GeneScreen nylon membrane (NEN Research Products, Boston, MA) in 0.4 M NaOH and hybridized with 32P-labeled mIFN-(3 cDNA fragment (Sen and Ransohoff,1993}.
Control mouse peritoneal macrophages constitutively expressed very low levels of IFN-~
mRNA. A significant elevation of IFN-(3 mRNA was found in macrophages incubated for 8 h with 30 PFU/cell of AdSCMV-LacZ. The increased expression of mouse IFN-~i (mIFN-(3) was also observed in macrophages infected with AdSCMV-mIFN HuIFN-j3, a replication-deficient adenoviral vector encoding human IFN-(3 gene, ruling out the possibility that the elevated expression of IFN-(3 gene was caused by a toxic effect of the LacZ gene.
IFN-~3 activity was determined in macrophages and NIH 3T3 cells. Macrophages or NIH 3T3 cells were incubated in medium or medium containing 30 PFU/cell AdSCMV-LacZ.
Culture supernatants were collected and irradiated on ice under a UV light for 30 min to inactivate the virus. To evaluate IFN-(3 activity, macrophages were plated into 96-well plates at a density of 105 cell/38-mm2 well and incubated with the samyles or increasing concentrations of IFN-~i (Lee BioMolecular, San Diego, California) in the presence of 1 mg/ml LPS (E coli, O111:B4, Sigma, St.
Louis, Missouri) for 24 h. N02 in the culture supc;matants was measured as described previously {bong et al., 1994b). A rat monoclonal antibody against mIFN-(3 (Yamasa Shoru Co., Tokyo, Japan) was added to the assay to confirm that the IFN activity detected was IFN-~3.

IFN-(3 activity was not detected in culture supernatants of control macrophages, whereas in macrophages infected with 30 PFU/cell of AdSCMV-LacZ for 2, 24, and 48 h, the supernatants contained 24 ~ 5, 112 t 20, and 67 ~ 20 U/106 macrophages, respectively. The production of IFN-(3 by the macrophages was transient and ceased after removal of AdSCMV-S LacZ. In contrast, neither control nor AdSCMV-LacZ-infected NIH 3T3 cells expressed IFN-~3 on the mRNA (RT-PCRTM analysis) or protein (activity) level.

Neutralization of Endogenous IFN-j3 Increases Expression of a Trans~' In the next set of studies, macrophages and NIH 3T3 cells were incubated with increasing concentrations of AdSCMV-LacZ, and (3-gal activity was determined 48 h later.
The cultures in 96-well plates were rinsed twice with PBS and lysed with 80 ml/well of lysis buffer (23 mM
NaH2P04, 77 mM Na2HP04, 0.1 mM MnCl2, 2 mM MgS04, 40 mM (3-mercaptoethanol, 0.1 Triton X-100, pH 7.3) at 37°C for 20 min. Twenty microliters of O-nitrophenyl-(3-D-galactopyranoside (ONPG) in warmed (37°C) substrate buffer (lysis buffer without Triton X-100) at 4 mg/ml was added to each well and allowed to react for 10 min (for NIH 3T3 cells) or 60 min (for macrophages). The reaction was stopped by the addition of 50 ml/well of 1 M Na2C03. The plates were read at 450 nm in a microplate reader (Dynach 5000). The (3-gal activity was calculated as U = (380 x A450)/10 or 60 (min) (MacGregoret al., 1991).
A rat monoclonal antibody against mIFN-~i or an equivalent amount (0.25 mg/ml) of rat IgG was added during the infection period at 10 neutralizing units (NU)/ml, which was sufficient to neutralize 100 IU/ml of IFN-~3 activity. At all concentrations of the vector, the anti-IFN-(3 antibody, but not the control, nonspecific IgG, increased LacZ gene expression threefold (FIG. 1 A).
The effect of the anti-IFN-(3 antibody was dose dependent, beginning at 1 NU/ml and reaching a plateau at 20 NU/ml (FIG. 1B). (3-gal activity was significantly increased in AdSCMV-LacZ-infected NIH 3T3 cells (FIG. 1 C) and was not altered by the presence of the anti-IFN-(3 antibody.
Even in the presence of the optimal concentration (20 NU/ml) of anti-IFN-~3 antibody, (3-gal activity was higher in the NIH 3T3 cells than in macrophages. Whether this difference was due to fewer adenovirus receptors on macrophages (Chu et al., 1992) or to inhibition in macrophages of viral infection by generation of IFN-a (Gessani et al., 1987) or nitric oxide (NO) (Croen, 1993;
Karupiah et al., 1993; Melkova and Esteban, 1995) is unclear.
Next, the inventors determined whether the continuous presence of the antibody was required to maintain enhanced expression of the transgene. Macrophages were infected with AdSCMV-LacZ in the presence of anti-IFN-/3 antiibody. Two days later, the macrophages were incubated in medium alone or medium containing 10 NU/mI of anti-IFN-(3 antibody. Macrophages infected in the presence of the antibody expressed higher levels of (3-gal activity than control macrophages. The (3-gal activity was maintained for up to 7 days in these macrophages regardless of whether they were subsequently cultured with or without anti-IFN-(3 antibody. Moreover, the addition of fresh antibody after the initial infection F~eriod did not further increase ~i-gal activity.
After their infection with AdSCMV-LacZ, analysis of ~i-gal activity in single cells was performed by flow cytometry (Fluorescence-Activated Cell Sorting; FACS).
Macrophages were cultured for 48 h with constant rocking. After a wash, the (3-gal activity was determined using a FluoReporter LacZ Flow Cytometry kit (Molecular Probes, Inc., Eugene, Oregon) following manufacturer's instructions. The analysis of (3-gal activity revealed two overlapping populations of macrophages. The majority (75%) of macrophages infected in the absence of the antibody were weakly fluorescent, with a mean relative fluorescence intensity (RFI) of 3.2;
25% exhibited stronger (RFI=22) ~i-gal activity. Similar results were obtained with macrophages infected in the presence of nonspecific rat IgG. The fluorescence intensity of macrophages infected with AdSCMV-LacZ in the presence of the anti-IFN-~i antibody was significantly higher, with 75% of the cells having an RFI of 15 and 25% having an RF~ I of 95 (FIG. 2).

Suppression of Transduction by Exogenous IFN-Q
Infection of NIH 3T3 cells with AdSCM'~-LacZ did not generate expression of IFN-(3 (Example 1 ). The inventors therefore analyzed whether the addition of exogenous IFN-(3 suppressed transduction efficiency. Indeed, the addition of 1 to 1000 U/ml mIFN-(3 to NIH 3T3 cells during infection with AdSCMV-LacZ produced a significant dose-dependent reduction in expression of ~3-gal activity (FIG. 3A). This inhibition was blocked by anti-IFN-~3 antibody but not by IgG (FIG. 3A).
Because macrophages respond to infection of AdSCMV-LacZ by producing IFN-(3, the S inventors next investigated whether macrophages (which are relatively resistant to infection) could affect AdSCMV-LacZ-mediated transduction of NIH 3T3 cells. NIH 3T3 cells were plated alone or onto monolayers of macrophages (1:1 ratio) and infected with the vector in the presence of rat IgG (control) or anti-IFN-(3 antibody. (3-gal activity in control cocultures (incubated in medium or with rat IgG) was ~50% lower than that in NIH 3T3 cells cultured alone (FIG.
3B). Infection of the cocultures in the presence of anti-IFN-(3 antibody partially prevented this inhibition (P<0.05) (FIG. 3B).

Production of Metalloelastase by Murine PEM
Specific pathogen-free female C57BL/6 mice were purchased from the Jackson Laboratory (Bar Harbor, ME). The mice were used when 8 to 12 weeks of age according to institutional guidelines. Animals were maintained in facilities approved by the American Association for Accreditation of Laboratory Animal Care and in accordance with current regulations and standards of the U.S. Department of Agriculture, Department of Health and Human Sciences, and National Institutes of Health.
PEM were collected by peritoneal lavage with HBSS from mice given an imraperitoneal injection of 1.5 ml of thioglycollate broth (Baltimore Biological Laboratories, Cockeysville, MD) 4 days previously (Saiki and Fidler, 1985). The PEMs were pelleted and resuspended in Eagle's MEM without serum, and 3 x 105 cells in 0.5 ml MEM were plated in 48-well cell culture clusters (Costar Co., Cambridge, MA). After 2 h, the wells were washed with EMEM to remove nonadherent cells. The resultant macrophage monolayer was >98% pure according to morphologic and phagocytic criteria. These cultures were then immediately treated as described below.

Murine PEM were plated for 2 h in serum-:free medium and then all nonadherent cells were removed. Serum-free DMEM-F 12 medium was added to the cultures. Forty-eight hours later, the culture supernatants were collected and treated for 20 rnin with the metal ion chelators EDTA (2 mM), EGTA (2 mM), or 1, I 0-phenanthraline ( 1 mM), which are potent inhibitors of MMPs (Barrett, I 977; Knight, 1977); with the serine protease inhibitors PMSF
( 10 mM), aprotinin ( 100 ~,g/ml), or trypsin inhibitor ( I 00 pg/ml) (Banda et al., 1987); or with the cysteinyl-proteinase inhibitor leupeptin ( 100 ~g/ml ) (Barrett, 1994).
Aliquots of control and treated supernatants were collected and spun at 3,000 x g at 4°C for 30 min to remove cell debris, and then added to 3H-labeled elastin. The elastase activity was determined 18 h later.
Elastase activity was assayed by a method described previously (Banda et al., 1987).
Briefly, bovine ligament elastin was radiolabeled with [3HJsodium borohydride (ICN) as described previously. Aliquots (0.1 ml) of serum-free conditioned medium were added to 200 pg of insoluble 3H-labeled elastin in 0.1 M of Tris-FICI (pH 8.0), 5 mM CaCl2 and 166.6 ~.g/ml sodium dodecyl sulfate in a total volume of 0.3 ml. The reaction was carried out at 37°C for 18 h with constant shaking and terminated by centrifugation in a microcentrifuge at 10,000 x g for 3 min. Radioactivity in aliquots ( I 00 pl) of supernata~its (containing degraded 3H-labeled elastin) was then measured in a liquid scintillation counter (;Beckman). A unit of elastinolytic activity was defined as the amount of enzyme degrading 1 ~g of 3H-labeled elastin/h (Banda et al., 1987).
As shown in Table 4, control PEM produced 22.2 ~ 1.0 U of elastase activity/106 cells.
The metal ion chelators (EDTA, EGTA, and 1,10-phf;nanthraline) significantly inhibited elastase activity (p<0.001 ), whereas the serine proteinase inhibitors (PMSF, aprotinin, and trypsin inhibitor) and cysteinyl-proteinase inhibitor (leupeptin) did not. These data confirm that murine PEM produce elastase (Werb and Gordon, 1975; 'White et al., 1977) which is classified as metalloproteinase (Shapiro, 1994; Senior et al., 1989; Banda and Werb, 1981 ).

Production of Metalloelastase by Murine PEM
Protease inhibitors Concentration Elastas 6 activity percent inhibitionb (U/IO cells) Medium control 22.2 ~ 1.0 EDTA 2 mM 0.7 t 0.1 96.8 EGTA 2 mM 1.4 ~ 0.2 93.7 1,10-Phenanthraline1 mM 1.3 ~ 0.1 93.9 PMSF 10 mM 24.6 ~ 2.3 0.0 Aprotinin 100 mg/ml 23.1 ~ 1.9 0.0 Trypsin inhibitor100 mg/ml 19.8 ~ 2.1 10.8 Leupeptin 100 mg/ml 22.7 ~ 2.6 0.0 aPEM culture supernatant was incubated 20 min with different concentrations of the indicated protease inhibitors prior to the addition of radiolabeled elastin substrate.
bPercent inhibition of elastase activity in comparison to control supernatants.
p<0.001.

Treatment of PEM with IFN-y and LPS Suppresses Elastase Production The inventors determined whether LPS and IFN-y, which activate PEM to become tumoricidal (Saiki and Fidler, 1985), would also regulate the production of elastase. PEM were incubated in serum-free DMEM-F 12 medium (control) or medium containing LPS ( 100 ng/ml), IFN-y ( 100 U/ml), or LPS ( 100 ng/ml) plus IFN-y ( 100 U/ml). At different times, elastinolytic activity in the culture supernatants was determined by the degradation of 3H-labeled elastin (FIG. 4). The production of elastase by control PEM was cumulative, reaching a peak by 48 h.
A significant reduction in elastase activity (p<0.001 ) was found in PEM
treated with LPS, IFN-y, or LPS plus IFN-y. These results indicate that tumoricidal activation of macrophages by LPS
plus IFN-y (Saiki and Fidler, 1985) does not correlate with enhanced elastase activity (FIG. 4).

Since the treatment of macrophages with LPS plus IFN-y induces the production of nitric oxide (Stuehr and Nathan, 1989), superoxide, and hydrogen peroxide (Nathan, 1982), which can interfere with cellular signaling pathways (Schreck et al., 1991; Moncade et al., 1991 ) and protein synthesis (Schreck et al., 1991 ), the inventors examined whether the inhibition of elastase in macrophages treated with LPS and IFN-y was associated with production of these free radicals. PEM were incubated with LPS ( 100 ng/rr~l), IFN-y ( 100 U/ml), or LPS ( 100 ng/ml) plus IFN-y ( 100 U/ml) in the absence or presence of 1 mM of the specific inducible nitric oxide inhibitor N-methyl arginine (NMA) (Stuehr and Griffith, 1992; Hibbs et al., 1987).
IO Nitrite concentration in culture supernatants was determined by a micropiate assay as described by Ding et al. (1988). Briefly, 50-p.l samples harvested from PEM-conditioned medium were treated with an equal volume of Gniess reagent ( 1 % sulfanilamide - 0.1 naphthylethylene diamine dihydrochloride - 2.5% H,P04) at room temperature for 10 min. The absorbance at 540 nm was monitored with a mic~roplate reader. Nitrite concentration was determined by using sodium nitrite as a standard.
NMA significantly inhibited production of NO measured as the level of nitrites (Senior et al., 1991 ) in the culture supernatants (FIG. SA) but did not prevent the inhibition of elastase activity (FIG. SB). Similarly, treatment of PEM vvith optimal concentrations of the oxygen radical scavengers superoxide dismutase or catalase (Blake et al., 1987;
Cheeseman and Slater, 1993) did not abrogate the inhibition of elastase acti~rity subsequent to treatment with LPS plus IFN-y (Table 5). Collectively, these data demonstratf; that LPS and IFN-y inhibit the production of elastase in PEM by a mechanism that is independent of nitric oxide, superoxide, and hydrogen peroxide.

Inhibition of Elastase Activity by LPS and IFN-'y is Independent of Superoxide and Hydrogen Peroxide Elastase activity (U/106 cells) Superoxide dismutase (U/ml) Catalase (U/ml) Treatments Medium 100 300 100 1000 None 18.211.8 16.411.9 15.81.6 19.52.1 14.02.1 LPS 3.0~0.3b 2.9t0.7b 1.8~0.8b 1.9f0.2b 1.8~O.Sb IFN-g 1.8~0.3b 1.9~0.4b 2.1~O.Sb 1.8~0.4b 1.7~O.Sb LPS+IFN-g 2.2tO.Sb 2.5t0.3b 1.9t0.6b 2.1 ~O.Sb 2.0~0.4b aPEM were incubated in serum-free DMEM-F 12 alone or medium containing LPS ( ng/nl) or IFN-y ( 100 U/ml) with or without indicated concentrations of superoxide dismutase or catalase. The culture supernatants were collected after 48 h and assayed for elastinolytic activity as described in Materials and Methods. Values are mean ~ SD of triplicate cultures. This is a representative study of 2.
p<0.001.

Regulation of Elastase Activity in PEM by Cytokines The inventors determined whether the expression of elastase by macrophages can be regulated by cytokines. To do so, the inventors incubated PEM with serum-free medium (control) or medium containing different concentrations of cytokines for 48 h.
Culture supernatants were harvested at 3,000 x g at 4°C for 30 min to remove cell debris, and tested for elastinolytic activity. Treatment of marine PEM with 100 U/ml of recombinant mouse IFN-y significantly inhibited the elastase activity (98% inhibition, p<0,001). In contrast, treatment of PEM with different concentrations of IFN-13, IL-la, IL-113, IL-2, IL-4, IL-6, IL-8, IL-10, TNF-a, TGF-a, TGF-13, bFGF, and marine 3E (MCF-1) did not alter the production of elastase nor the viability of the macrophages.

The inventors next examined the effect of th~~ above cytokines on production of elastase by resident peritoneal macrophages. The resident macrophages secreted lower levels of elastase as compared to thioglycollate-elicited macrophages x;2.65 ~ 0.32 vs. 19.58 ~
1.23 U/ I 06 cells/48 h). Elastase activity was decreased in the resident macrophages by incubation with LPS and IFN-y. Unlike the inflammatory macrophages, the M-CSF and GM-CSF did not affect secretion of elastase by resident peritoneal macrophages.
Since colony-stimulating factors (CSF) have been shown to enhance the proliferation of macrophages and modulate their function (Brach and Herrman, 1991; Crosier and Clark, 1992;
Metcalf, 1990; Chen et al., 1988; Sampson-Johannes and Cerlino, 1988), the inventors examined whether treatment of PEM with G-CSF, M-CSF, or GM-CSF altered the production of elastase.
PEM were incubated in medium (control) or medium containing different concentrations of the CSFs for 48 h, and then the culture supernatants were collected at 3,000 x g at 4°C for 30 min to remove cell debris and assayed for elastinolytic activity. Treatment of PEM
with GM-CSF
enhanced elastase production in a dose-dependent manner (FIG. 6A), whereas treatment of PEM
with M-CSF inhibited it (FIG. 6B). G-CSF treatment did not affect elastase production (FIG. 6C).
In the next set of studies, the inventors examined whether the treatment of PEM with GM-CSF could abrogate (or reverse) the inhibition of elastase activity produced by LPS and IFN-y. As shown in Table 6, treatment of PEM with GM-CSF (10-1000 U/ml) increased the production of elastase from 11.3 U/ml to 32.9 U/ml hut did not prevent the inhibition of elastase activity in macrophages incubated with LPS and IFN-y.

WO 98/22605 PCT/US97t21475 Elastase Activity in PEM Incubated With GM-CSF in the Presence of LPS and IFN
y Elastase activity (U/106 cells) 0 U/m! 10 U/ml 100 U/ml 1000 U/ml Treatment9 GM-CSF GM-CSF GM-CSF GM-CSF

Medium 11.3 ~ 0.9 14.91 1.7 21.1 ~ 1.8 32.9 ~ 2.3 LPS 2.0 ~ 0.4b 2.4 ~ 0.4b 4.3 t 0.7b 6.9 t 0.9b IFN-y 3.8~0.3b 5.7~0.9b 10.40.8 14.9 1.3 LPS+IFN-y 2.2t0.3b 2.4t0.5b 2.8~0.9b 5.0~0.7b aPEM were incubated in serum-free DMEM-F 12 alone or medium containing LPS ( ng/ml) or IFN-y ( 100 U/ml) with different concentrations of recombinant GM-CSF. The culture supernatants were collected after 48 h and assayed for elastinolytic activity as described in Materials and Methods. Values are the mean of elastase activity (U/106 cells) ~ S.D. of triplicate cultures. This is a representative study of 3.
p<0.001.
Elastolytic activity was also determined by K-elastin zymography (Senior et al., 1991 ).
Briefly, aliquots of conditioned medium from control and test PEM were subjected to substrate gel electrophoresis. PEM were incubated in DMEM-F 12 alone or with LPS ( 100 ng/ml), IFN-y (100 U/ml), GM-CSF (1000 U/ml), M-CSF (10 ng/ml), or G-CSF (1000 U/ml). The supernatants were collected after 48 h, concentrated, and analyzed by elastin zymography. The samples were applied without reduction to a 12% SDS-polyacrylamide gel impregnated with 1 mg/ml K-elastin (ETNA-elastin, Elastin Products, Owensville, MO). Following electrophoresis, the gels were washed twice for 15 min each time in washing buffer (50 mM Tris, pH 7.5, 5 mM
CaCl2, 1 ~,M ZnCl2, 2.5% Triton X-100), and then incubated at 37°C for 48 h with shaking in the buffer that also contained 1 % Triton X-100. The gels were stained with a solution of 0.1 Coomassie brilliant blue R-250. Elastolytic activity was indicated by the appearance of a clear zone of lysis in a blue background. Molecular weight of the elastolytic bands were estimated by using prestained molecular weight markers (BioRad).

Macrophages secrete 22-kDa active MME. The high molecular weight forms may represent differentially cleaved fragments of the 5_S-kDa proenzyme (Sampson-Johannes and Cerlino, 1988). As revealed by zymogram, the changes in the elastolytic activity of macrophages treated with LPS, IFN-y, GM-CSF, or G-CSF resulted from alteration in the level of metalloelastase protein.
EXAMPLE. 7 Northern Blot Anahrsis The inventors determined whether treatment: of PEM with various cytokines or LPS
regulated the expression of MME mRNA. PEM were incubated in medium alone or medium containing LPS ( 100 ng/ml), IFN-y { 100 U/ml), GM-(~SF ( 1000 U/ml), M-CSF ( 10 ng/ml), or G-CSF ( 1000 U/ml), at which point mRNA was isolated and analyzed for MME-specific mRNA
transcripts on northern blots. Using the Fast Track mRNA isolation kit (Invitrogen, San Diego, CA), polyadenylated mRNA was extracted from 5 x l0' PEM that had been cultured in medium alone or with different agents. mRNA (2.5 pg mRNA/lane) was electrophoresed on denaturing formaldehyde/agarose gel, electrotransferred at 0.6 A to a GeneScreen nylon membrane (DuPont Co., Boston, MA), and UV cross-linked with 120,000 p,J/cm2 using a UV
Stratalinker 1800 (Stratagene, La Jolla, CA). Hybridi.zations were performed with 1.7-kb cDNA
fragment corresponding to marine macrophage elastase (MME; Shapiro et al.) 1992), human TIMP-1 cDNA that detects a mouse 0.9 kb transcript (Campbell et al., 1987), and 1.3-kb gene fragment corresponding to rat GAPDH cDNA (F~rt et al., 1985) as described previously (Radinsky et al., 1987). Nylon filters were washed three times at 55-60°C with 30 mM NaCI, 3 mM sodium citrate (pH 7.2), and 0.1 % sodium dodecyl sulphate (w/v).
The cDNA probes used in this analysis were a 1.3-kb PstI cDNA fragment corresponding to rat GAPDH (Fort et al., 1985), a 1.7-kb BamH:f cDNA fragment of marine macrophage elastase (MME) (obtained from Dr. S. D. Shapiro, Sn. Louis, MO) (Shapiro et al., 1992), and a 0.63-kb EcoRI-KpnI gene fragment corresponding o~o metalloproteinase inhibitor-1 (TIMP-1 ) (obtained from Dr. W. Stetler-Stevenson, NIH, NCI, l3ethesda, MD). Each cDNA
fragment was purified by agarose gel electrophoresis, recovered using GeneClean (Bio 101, Inc., La Jolla, CA), and radiolabeled by the random primer technique using [a-32P]-dNTP (Feinberg and Vogelstein, 1983).
Control PEM expressed a specific mRNA transcript for MME. Treatment of PEM
with LPS or IFN-y inhibited steady-state expression of MME mRNA by 70% and 80%, respectively.
Treatment of PEM with GM-CSF increased expression of MME mRNA (40%), whereas M-CSF
decreased it by 70% and G-CSF had little to no effect.
Since elastase activity is dependent on the balance between production of elastase and TIMP-1 (Campbell et al., 1987; Shapiro et al., 1992; Welgus et al., 1992;
Docherty et al., 1985), the inventors examined the steady-state expression of TIMP-1 mRNA in PEM.
Normal (untreated) PEM did not express TIMP-l, whereas PEM treated with LPS and M-CSF
demonstrated a 6.1- and S.1-fold increase in TIMP-1-specific mRNA transcripts, respectively.
Treatment of PEM with IFN-y, GM-CSF, or G-CSF slightly increased the steady-state level of TIMP-1 mRNA. Collectively, these data show that the expression of TIMP-1 by macrophages is differentially regulated by different CSFs and suggest a potential mechanism by which treatment of PEM with M-CSF would inhibit elastase activity.

Upregulation of MME Expression The inventors determined whether the upregulation of steady-state mRNA was due to increased transcription or enhanced stability of the message (Sessa et al., 1992; Weisz et al., 1994; Vodvotz et al., 1993). The inventors carried out nuclear run-on assay using nuclei isolated from inflammatory macrophages. PEM (5 x 10') were cultured 4 h in medium (control) or medium containing GM-CSF (1000 U/ml), and were then harvested. Cells were then lysed, nuclei were isolated and incubated with a transcription buffer containing a 32P-labeled UTP to label nascent RNA molecules as previously described (Sessa et al., 1992; Weisz et al., 1994) with modifications. After in vitro transcription, nuclear RNA was isolated using TRI reagent (Molecular Research, Inc., Cincinnati, OH), and hybridized to immobilized DNA
probes.
Following hybridization, filters were washed and autoradiographed.

32P-labeled RNA was normalized to the lowest sample to achieve 5 x 105 cpm/ml of hybridization buffer consisting of 10 mM Tris, pH 7.4, 10 mM EDTA, 0.2% SDS, 300 mM
NaCI, 0.1 % sodium pyrophosphate, 1 x Denhardt's solution, and 100 p,g/ml salmon sperm DNA.
For hybridization, 10 ~g of a marine MME cDNA- containing plasmid and a 10 ~g of a rat S GAPDH cDNA containing plasmid were denatured and dot blotted on GeneScreen membrane (DuPont, Boston, MA). After 72 h hybridization, the filters were washed in 2x SSC at 65°C, and then in 2x SSC containing 0.1% SDS at 65°C. Air-dried filters were exposed to X-ray film. The relative transcriptional rate was determined as a ratio of the average area of MME-specific signal and GAPDH-specific signal using a personal densitometer {Molecular Dynamics, Sunnyvale, CA).
Expression of the MME gene was quantified by densitometry of autoradiograms using Image Quant software program (Molecular Dynamics, Sunnyvale, CA). The value for each sample was calculated as the ratio of the average areas of MME-specific mRNA
transcripts to the 1.3-kb GAPDH mRNA transcript in the linear range of the film. The rate of in vitro transcription determined by densitometric analysis of autoradiograms demonstrated that GM-CSF induced the transcription of the MME gene (30%) as compared to the control.
To determine whether GM-CSF affected the stability of MME mRNA transcripts, PEM
were incubated for 18 h in medium alone or medium containing 1000 U/ml GM-CSF.
The cells were washed and de novo synthesis of RNA was inhibited by 10 pg/ml actinomycin-D (Docherty et al., 198S). RNA was extracted at different time points and separated on 1 %
agarose, transferred onto a nylon membrane, and probed with 32P-labeled MME and GAPDH
cDNA
probes. As shown in FIG. 7, treatment of macrophages with GM-CSF resulted in stabilization of 2S MME mRNA as compared to the medium control.
EXAMPLE; 9 Resection of Local Subcutaneous Tumors and Outgrowth of Lung Metastases The inventors determined whether the excision of a local ("primary") subcutaneous 3LL
carcinoma would enhance the growth of spontaneovus lung metastases. CS7BL/6 mice (8-12 weeks old) were implanted s.c. with cells from 3LL-met (metastatic) or 3LL-nm (nonmetastatic) variants. The nonmetastatic variant 3LL-run was isolated from a 3LL tumor originally obtained from the National Cancer Institute Tumor Bank (NCI-Frederick Cancer Research Facility, Frederick, MD) (Talmadge and Fidler, 1982). The metastatic variant 3LL-met was obtained from Dr. M. O'Reilly (Harvard Medical School, Boston, MA) (O'Reilly et al., 1994). Both tumor cell lines were maintained in tissue culture in Eagle's minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, nonessential amino acids, L-glutamine, and a twofold vitamin solution. Adherent cultures were maintained on plastic and were incubated in 5% C02-95% air at 37°C. The cultures were free of Mycoplasma and pathogenic mouse viruses.
Subcutaneous tumors (8-12 mm in diameter) were resected aseptically. All necrotic zones were removed and the viable tissue was minced and dissociated with collagenase (Type I, 200 Ulml) and DNase (270 U/ml) (Sigma Chemical Co., St. Louis, MO) as described previously (Dong et al. , 1994a). Cells were suspended in MEM containing 10% FB S and plated at 5-10 x 106 viable cells/T150 flask. After a 3-hr adherence, the cultures were rinsed and given fresh medium. Forty-eight hours later, the adherent tumor cells were harvested by brief trypsinization, washed in MEM-10% FBS, and resuspended in Hanks' balanced salt solution (HBSS). Aliquots of 106 cells in 0.1 ml of HBSS were injected into the dorsal subcutis in the proximal midline.
When tumors were 12-15 mm in diameter, the mice were anesthetized with methoxyflurane. The tumors in one group of mice were surgically excised, and the area closed with metal wound clips.
The other group underwent a sham surgical procedure, which left the s.c.
tumors intact. The mice were monitored daily and killed 10-14 days after surgery. The lungs were weighed and fixed in Bouin's solution. The tumor nodules were counted under a dissecting microscope.
Although the two variant 3LL carcinoma cell lines had similar growth patterns in culture, the 3LL-met produced rapidly growing tumors, and the 3LL-nm cells produced slow-growing tumors in the dorsal subcutis of syngeneic mice. Specifically, the s.c. tumors reached 12-15 mm in diameter on days 14 and 28 for the 3LL-met and 3LL-nm cells, respectively.
The 3LL-run tumors did not produce visible lung metastases regardless of whether the local s.c. tumor was resected or not (even after 60 days). In contrast, on day 28 of the study, the 3LL-met s.c. tumors produced a median of 10 lung metastases in mice with progressively growing s.c. tumors. In mice with resected s.c. tumor, the median number of visible lung metastases was 61 (P>0.05), and the size of the lung metastases was significantly enhanced (P<0.01 ) (Table 7). These results show that the surgical resection of a metastatic variant of the 3LL tumor significantly enhances the size of spontaneous lung metastases.
TABLE T
Enhanced Growth of Spontaneous 3LL Lung Metastases in Mice Subsequent to Resection of the Primary Subcutaneous Tumor Lung Metastases Lung Weight Cell Lines Treatment Median Range (mg) 3LL-nm Sham surgery 0 - 200 ~ 16 Resected 0 - 205 ~ 18 3LL-met Sham surgery I 0 0-90 251 ~ 14 Resected 61 a 34-145 474 ~ 29b C57BL/6 mice were injected s.c. (dorsal proximal midline) with 1 x 106 3LL
cells (derived from s.c. tumors). When the tumors reached 12-15 mm in diameter (10-14 days for 3LL-met and 24-28 days for 3LL-nm cells), the mice were anesthetized. The s.c. tumors were resected from one group of mice (n=10). The other group underwent sham-surgery. The mice were killed 2 weeks later.
The Iungs were weighed and fixed in Bouin's solution, and spontaneous metastases were counted under a dissecting microscope.
I 5 aP>0.05.
bP<0.01.

Macrophage Infiltration into s c Tumors and Exuression of Metalloproteinases Macrophages in 3LL tumors or in cultures established from these tumors were identified by immunohistochemistry using the macrophage-specific F4/80 antibody (Austyn.ar~d Gordon, 1981 ). Subcutaneous tumors (8-10 mm in diameter) were resected and the tumor samples were fixed in formalin for hematoxylin and eosin staining or in liquid nitrogen for immunohistochemical staining using F4/80. Sections (8-10 mm) of frozen tumor tissue or cultured cells fixed with 0.125% glutaraldehyde in PBS were treated with a rat monoclonal F4/80 antibody followed by gold-labeled secondary antibody as described in detail previously (Bucana et al. , 1992).
Immunohistochemistry staining of cryostat sections using marine macrophage-specific antibody F4/80 revealed that both 3LL-nonmetastatic (nm) and 3LL-metastatic (met) s.c. tumors were infiltrated by macrophages; the density in the 3LL-nm tumors was higher.
A similar pattern of staining was observed using antibody against scavenger receptor (Fraser et al. , 1993 ), which also identifies macrophages.
Next, the expression of MMPs in the 3LL s.c. tumors and their cultured cells was examined. The mRNA was extracted from tumor tissue or cell cultures (>3 passages in culture) using the Fast-Track kit (Invitrogen, San Diego, CA). For Northern blot analysis, 1 p,g/lane of mRNA was fractionated on 1 % denaturing formaldehyde/agarose gels, electrotransferred to GeneScreen nylon membrane (DuPont Co., Boston, MA), and UV cross-linked with 120,000 mJ/cm2 using a UV Stratalinker 1800 (Stratagene, La Jolla, CA). Hybridizations using cDNA
probes were performed as previously described (Dong et al. , 1994a). The filters were washed two or three times at 50-60°C with 30 mM NaCI/3 mM sodium citrate, pH
7.2/0.1 % sodium dodecyl sulfate. The DNA probes used were cDNA fragments corresponding to rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Dong et al., 1994b), MMP-2 (ATCC, Rockville, MD), MMP-9 (Levy et al., 1991 ), and MME (Shapiro et al., 1992).
mRNA
expression was quantified on an LKB Ultrascan XL laser densitometer (Pharmacia LKB
Biotechnology, Uppsala, Sweden). Each sample measurement was calculated as the ratio of the average area of the proteinase transcripts to that of the GAPDH transcript.
The quantitative analysis of proteinase expression is summarized in Table 8.

Expression of Proteinase mRNA Transcripts in 3LL Tumors and Cultured Cells MMP mRNA 3LL-nm 3LL-met (MMP/GAPDI-n Tumor Cell Culture: Tumor Cell Culture MME 339a 1 121 1 aT'he level of expression of the MMP genes vvas quantitated by densitometry readings of autoradiograms using the ImageQuant software program (Molecular Dynamics).
Each sample measurement was expressed as the ratio of the average; area under the curve of the specific mRNA
transcripts to 1.3-kb GAPDH mRNA transcripts.
High levels of MME mRNA were detected in both 3LL-nm and 3LL-met tumor tissues, low Levels were detected in the 3LL cells cultured in vitro. Correlated with the extent of macrophage infiltration, the 3LL-nm tumors expre;csed a significantly higher level of MME
mRNA than did the 3LL-met tumors. The 3LL-nrn tumors also expressed a higher level of MMP-9 than did the 3LL-met tumors. Neither 3LL-run nor 3LL-met cultures expressed MMP-9 transcripts. 3LL-nm and 3LL-met s.c. tumors expressed similar levels of MMP-2 mRNAs but only 3LL-met cells expressed MMP-2 mRNA in culW re.

Expression of MME and Generation of Angiostatin The above data indicate that the predominant: proteinase in the 3LL tumors was MME.
Since elastase has been shown to cleave plasminogen to fragments that include angiostatin (O'Reilly, et al., 1994), the inventors next determined whether the MME in the 3LL tumors is associated with generation of angiostatin activity. (:ells isolated by enzymatic dissociation of 3LL-met tumors were cultured (as in Example 9 above). Immediately after dissociation and subsequent to a different number of passages in culh~re (to deplete macrophages), the inventors determined macrophage content (immunohistochemi stry staining with the macrophage specific F4/80 antibody; see Example 10 above), MME mRNA (northern blot as in Example 10 above), MME activity in the culture supernatant (enzymatic assay), and generation of angiostatin subsequent to the addition of plasminogen (bioassay).
Elastase activity in the conditioned medium was determined by a method described previously using [3H)-NaBH4-reduced elastin as a substrate (Werb et al., 1986). The samples were mixed in reaction buffer { 100 mM Tris/HCI, 5 mM CaCl2, 0.2 mg/ml SDS and 0.006%
NaH3) and incubated at a concentration of 600 mglml at 37°C for 16 hr.
Free-form 3H release was monitored, and the enzyme activity was expressed as cpm/reaction.
Angiostatin activity, i. e. , inhibition of endothelial cell proliferation, was determined as described previously (O'Reilly et al., 1994). Briefly, bovine capillary endothelial cells {BCE), obtained from Dr. O'Reilly {Harvard Medical School, Boston, MA) were seeded onto gelatinized 24-well plates at 1.25 x 104/well/0.5 ml DMEM-10% calf serum (CS) supplemented with 3 ng/ml of human bFGF (Genzyme, Cambridge, MA) in 10% C02 (O'Reilly et al., 1994). The cells were allowed to adhere overnight, rinsed, and incubated with 0.25 ml/well DMEM-5% CS
or test samples for 20 min. Additional medium containing bFGF was then added to a final concentration of 1 ng/ml at 0.5 ml/well. Seventy-two hours later, the cells were harvested by trypsinization and counted. Angiostatin activity was expressed as percent inhibition of endothelial cell growth in culture.
The data shown in FIG. 8A, FIG. 8B, FIG. 8C and FIG. 8D demonstrate that, with increasing passage number, the number (content) of macrophages decreased, and after three passages in vitro, the inventors found no evidence of macrophages among the 3LL cells (FIG. 8A). The level of MME transcript (FIG. 8B) and elastase activity (FIG.
8C) directly correlated with the number of macrophages in the cultures. Human plasminogen was added to the cultures (at different passages). The supernatant fluids were recovered 72 hr later and tested for angiostatin activity. The highest angiostatin activity was found in cultures containing the highest number of macrophages (immediate cultures, passage 0) and declined with increasing passage number. No angiostatin activity was found in cultures depleted of macrophages or in the absence of plasminogen (FIG. 8D). The angiostatin activity (antiproliferation) was specific for the endothelial cells: It did not inhibit division of 3LL cells, B 16 melanoma cells, or NIH-3T3 cells, agreeing with a published report (O'Reilly et al., 1994).

The elastinolytic activity detected in the 3LL-met tumor was a MME. The inventors base this conclusion on the results of studies using various selective proteinase inhibitors (Table 9).

Identification of Elastase Derived from 3LL Tumor Tissue as Metalloproteinase 1.10-Phenanthraline PMSF
Samples Control 1 mM 10 mM 1 mM 10 mM
3LL tumor tissue 8383 ~ 144a 57 ~ 44b Ob 8324 ~ 334 9758 ~ 937 (200 mg/ml) Macrophage supernatant 4084 ~ 47 1070 ~ 11846 Ob 3277 ~ 84 4637 ~ 115 Pancreatic elastase 3497 ~ 96 3277 ~ 84 463 7 ~ 115 698 t 2146 440 ~ 327b aFree 3H released from the labeled elastin in the reaction.
bP<0.001.
At 10 mM, the serine proteinase inhibitor PM:SF produced >80% inhibition of pancreatic serine elastase activity, but it did not inhibit the elastinolytic activity from the 3LL tumor tissue or from purified cultures of macrophages. In contrast, elastase activity from both the tumor tissue and macrophages, but not the pancreatic elastase, was completely suppressed by the MMP
inhibitor 1,10-phenanthraline.

Secretion of Elastase and Generation of Angiostatin in Cocultures of 3LL Cells and Macrophag-eses To determine whether 3LL cells can regulate elastase activity and hence regulate production of angiostatin in macrophages, the inventors next cultured purified peritoneal exudate macrophages in serum-free medium with or without 3LL-met or 3LL-nm cells in the absence (FIG. 9A) or presence (FIG. 9B) of plasminogen. PEM were collected by peritoneal lavage with HBSS from mice given an intraperitoneal (i.p.) injection of 1.5 ml of thioglycollate broth 4 days previously. The PEM were plated onto 24-well dishes at a density of 2 x 105 cellslwell. After 2 hr, the adherent cultures were washed. The resultant PEM population was >98%
pure according to morphologic and phagocytic criteria (Dong et al., 1993a).
PEM were incubated for 24 hr with tumor cells (2 x 105/well) or with tumor cell conditioned medium in MEM-5% FBS. The cultures were washed, and the cells were incubated in 0.5 ml serum-free DMEM-F 12 medium (for elastase assay) or medium containing 200 mg/ml plasminogen for 72 hr (for angiostatin activity assay). The medium was collected and centrifuged at 3000 g at 4°C for 30 min. The supernatants were used in the assays as described in Example 11.
In agreement with a previous report (Werb and Gordon, 1975), the inflammatory macrophages constitutively secreted elastase (FIG. 9A) and expressed MME mRNA, as determined by northern blot analysis. A detectable (baseline) level of elastinolytic activity was detected in culture supernatants of 3LL-met and 3LL-nm cells. Since the 3LL
cells did not express MME mRNA, the inventors attribute the elastinolytic activity to other proteases released by the tumor cells. Supernatants of macrophage-3LL cocultures contained a 3-fold higher elastinolytic activity than supernatants harvested ftom macrophages cultured alone (FIG. 9A). In parallel studies, the inventors added plasminogen to the macrophages cultured alone, 3LL cells cultured alone, or macrophage-3LL cocultures. Angiostatin activity (inhibition of endothelial cell proliferation) directly correlated with elastinolytic activity:
macrophages cultured alone produced 24% inhibition of endothelial cell growth, whereas macrophages cultured with 3LL

cells produced 43-46% inhibition of endothelial cell growth. The angiostatin activity in supernatants of 3LL cells cultured alone was <4% (FIG. 9B).
To verify that the inhibition of endothelial cf:ll proliferation was due to angiostatin, the - 5 inventors identified this 38-kDa fragment of ~plasminogen (O'Reilly et al., 1994) by immunoblotting with a monoclonal antibody against lysine-binding site-1. The supernatants of macrophages plus plasminogen, macrophages and 3LL-met plus plasminogen, or 3LL-met plus plasminogen were mixed with sample buffer (62.5 mM Tris/HCI, pH 6.8, 2.3% SDS, 100 mM
DTT, and 0.05% bromophenol blue), boiled, and separated on 10% SDS-PAGE. The protein IO was transferred onto 0.45 mm nitrocellulose membranes. The filter was blocked with 3% BSA
in TBS (20 mM Tris/HCI, pH 7.5, 150 mM NaClj, probed with antibody against the lysine binding site I (LBS-1; 1 mg/ml) in TTBS (TBS containing 0.1 % Tween 20), incubated with a second antibody in the buffer, and visualized by the ECL Western blotting detection system (Dong et al., 1993a).
Consistent with the bioassay (FIG. 9B), the 38-kDa fragment was found in the supernatant of the macrophage-3LL cocultures incubated in medium plus plasminogen and, to a lesser extent, in the supernatants of macrophages incubated alone in medium containing plasminogen but not in culture supernatants from 3LL-met cells incubated in medium plus plasminogen.
To ascertain that the increased generation of angiostatin in the 3LL
macrophage cocultures was due to macrophage-derived MME, the inventors next incubated macrophages in medium (control) or medium conditioned by 3LL-mea cells in the presence or absence of human plasminogen. Elastinolytic and angiostatin activities were then determined (FIG. l0A and FIG. lOB). The data clearly demonstrate that incubation of macrophages with supernatants of the 3LL cultures significantly increased the secretion of elastase and the generation of angiostatin.

GM-CSF Released by 3LL Cells is Responsible for Increased Secretion of MME by Macrophages Since GM-CSF is the only cytokine (among many examined) that can upregulate MME
expression in murine macrophages (Example 8), the inventors next determined whether 3LL
cells produced and released GM-CSF and whether it in turn increased elastinoiytic activity in macrophages and, hence, production of angiostatin. Polyadenylated mRNA
extracted from subcutaneous 3LL-nm tumor, 3LL-nm cell culture, 3LL-met tumor and 3LL-met cell culture was treated with RQ1 RNase-free DNase and reverse transcribed using an AMV reverse transcriptase system (Promega). Resulting cDNA from 20 ng mRNA was amplified with 2.5 U of Taq polymerase in a reaction volume of 50 ml containing 10 mM Tris-HCI, pH 9.0 at 25°C, 1.5 mM
MgCl2, 50 mM KCI, 0.1% Triton X-100, 200 mM concentrations of each of the dNTPs, and 200 nM concentrations each primer (primer number 1, CCCATCACTGTCACCCGGCCTTGG (SEQ
ID N0:5) and primer number 2, GTCCGTTTCCGGAGTTGGGGGGC (SEQ ID N0:6), defining a 279-by fragment of GM-CSF; and primer number 3, GTGGGCCGCTCTAGGCACCA (SEQ ID N0:3) and primer number 4, CGGTTGGCCTTAGGGGTCAGGCTGG (SEQ ID N0:4), defining a 245-by fragment of (3-actin [Stratagene, La Jolla, CA]). Amplification was carried out on a Perkin-Elmer-Cetus thermal cycler for 25 cycles ( 1 cycle = 94°C, 45 sec; 60°C, 45 sec; and 72°C, 1 min). Thirty-microliter aliquots of each resulting mixture were separated on 1.5% agarose gel and visualized under ultraviolet light.
RT-PCRTM analysis showed the presence of GM-CSF mRNA in both the 3LL-nm and 3LL-met variants growing in culture. Similar levels of steady-state GM-CSF
mRNA were also found in the s.c. tumors. Culture supernatants of 3LL-met and 3LL-nm variants contained 28 pg and 40 pg/106/72 hr of GM-CSF, respectively. Further evidence of the role of GM-CSF
(released by 3LL cells) in the regulation of MME and production of angiostatin activity came from neutralization studies (FIG. 11 A and FIG. 11 B). Murine macrophages were incubated with medium (control) or medium conditioned by 3LL-nm or 3LL-met cells in the absence of presence of antibodies against mouse GM-CSF or control-nonspecific IgG.
Antibodies against GM-CSF but not those against IgG significantly decreased production of elastase (FIG. 11 A) and angiostatin activity (FIG. 11 B). Neither the anti-(iM-CSF antibody nor the nonspecific IgG
affected the generation of MME or angiostatin activity in macrophages incubated in medium alone.
EXAMPLE,14 Expression of MMP-9 is Not Reguired for Production of Angiostatin MMP-9 is constitutively released from marine macrophages, and its secretion can be increased by a variety of stimuli (Xie et al., 1994). Since MMP-9 has some elastinolytic activity (Senior et al., 1991 ), the inventors wished to determine whether it contributed to the generation of angiostatin in the 3LL carcinoma-macrophage system. The inventors therefore cultured macrophages in medium alone (control) or medium containing 1 mg/ml LPS or 1000 U/ml GM-CSF in the absence (for enzyme assays) or presence (for production of angiostatin) of 200 mg/ml plasminogen. Elastase and angiostatin activities were determined as described in Example 11 above. MMP-9 activity was determined by gelatin zymography.
Aliquots of conditioned medium from control and test macrophages were subjected to substrate gel electrophoresis in a 7.5% polyacrylaJnide slab gel impregnated with 2 mg/ml gelatin (Sigma). After electrophoresis, the gel was washed for 30 min at room temperature in washing buffer (50 mM Tris-HCI, pH 7.5, 5 mM Ca.Cl2, 1 mM ZnCl2, 2.5% Triton X-100) and then incubated for 24 hr at 37°C with the washing buffer containing 1 %
Triton X-100. The gel was stained with Coomassie Blue 8250. The gelatinolytic activity was quantified using densitometric scanning (Molecular Dynamics, Sunnyvale, CA) (Xie et al., 1994).
As shown in Table 10, while LPS increased secretion of MMP-9, it decreased release of MME. Incubation of macrophages with GM-CSF enhanced secretion of MME without affecting the levels of MMP-9. Since generation of angiostatin was suppressed by LPS and augmented by GM-CSF, the inventors conclude that MME (and not MMP-9) was responsible for the generation of angiostatin by macrophages (Table 10).

Correlation of MME and MMP-9 Production by Macrophages with Generation of An~iostatin Macrophage Elastase MMP-9 Angiostatin Treatment Activity Activity Activity Medium 37.0 ~ 2.9 193 22.2 ~ 4.8~
LPS 6.5 ~ 2.6a 3896 8.9 ~ 7.1 GM-CSF 108.0 ~ 7.Sa 201 b 43.7 ~ 1.8°
aMME activity, cpm x 103/reaction.
bZymography, densitometry units.
~Angiostatin activity; percent inhibition of BCE growth.

Suppression of Metastases by Primary Tumors Engineered to Release GM-CSF
In a well-accepted animal model, the inventors show by this example effective suppression of angiogenesis, and correlating suppression of metastases in secondary tumor sites.
This suppression is achieved where GM-CSF released from a subcutaneous primary tumor recruits macrophages into the tumor lesion and stimulates MME expression in these tumor infiltrating macrophages. The MME in turn degrades plasminogen to angiostatin that circulates into distant capillary beds where angiogenesis and metastatic development is inhibited.
I. Materials and Methods Mice Specific pathogen-free male athymic nude mice were purchased from Jackson Laboratory, Bar Harbor, ME. The mice were maintained according to institutional guidelines under specific pathogen-free conditions in facilities approved by the American Association for Accreditation of .Laboratory Animal Care and in accordance with current regulations and standards of the Department of Agriculture, Department of Health and Human Services, and the National Institutes of Health. The mice were used when they were 8 to 12 weeks old.

Cell Culture Marine metastatic Lewis lung carcinoma (31:.L-met), obtained by the method described by O'Reilly et al., incorporated herein by reference (O'Reilly et al., 1994a);

(Dranoff, et al. , 1993 ), a variant of mouse B I 6-F 10 melanoma that was transduced by using a MFG retroviral vector encoding marine GM-CSF (obtained from Dr. D. Pardoll, John Hopkins University); K1735M2 melanoma cells (Staroselsky, et al., 1991); and renal carcinoma RENCA
cells (Dinney et al., 1991) were maintained in tissue culture in Eagle's minimal essential medium (MEM) supplemented with S-10% fetal bovine serum (FBS), sodium pyruvate, nonessential amino acids, L-glutamine, and a twofold vitamin solution. Adherent cultures were maintained on plastic and were incubated in 5% C02-95% air at 37°C. The cultures were free of Mycoplasma and pathogenic mouse viruses.
Marine K1735M2 melanoma cells were maintained in MEM supplemented with 5% FBS. The cells were transfected with the plasmid pcDNA3-GM-CSF driven by human cytomegalovirus promoter (obtained from Dr. W. gang, Academia Sinica, Taiwan) to derive KI735M2-GM cells or a control plasmid encoding neomycin resistance gene. The cells were then selected in 6418 for up to 3 weeks. Bovine capillary endothelial cells (BCE) obtained from Dr. O'Reilly (Harvard Medical School, Boston, MA) were cultured on gelatinized surfaces in DMEM-10% calf serum (CS) supplemented with 3 ng/ml human bFGF (Genzyme, Cambridge, MA) in 10% C02 (O'Reilly et al., 1994b).
Animal Studies Aliquots of B 16-F 10-GM or K 173 SM2-GM cells in 0.1 ml of HB S S were inj ected into the dorsal subcutis in the proximal midline. When tumors were 12-15 mm in diameter, the mice were anesthetized with methoxyflurane. The tumors in one group of mice were surgically excised, and the area closed with metal wound clips. The other group underwent a sham surgical procedure, which left the subcutaneous tumors intact. One day later, 105 of 3LL-met or K 173 SM2 cells were inj ected intravenously or 1 O5 o:f RENCA cells were inj ected subcapsularly into one kidney of the mice. The mice were killed a! 1-28 days (3LL-met) or 15 days (RENCA) later. The lungs or kidney were weighed and fixed in Bouin's solution. The tumor nodules in the lungs were counted under a dissecting microscope.

Collection and Cultivation of Mouse Peritoneal Macrophages (PEM) PEM were collected by peritoneal lavage with HBSS from mice given an intraperitoneal (i.p.) injection of 1.5 ml of thioglycollate broth 4 days previously. The PEM
were plated onto 24-well dishes at a density of 2 x 105 cells/well. After 2 hr, the adherent cultures were washed.
The resultant PEM population was > 98% pure according to morphologic and phagocytic criteria (Dong et al., 1993b).
Elastase and angiostatin.
PEM were incubated for 24 hr with tumor cells (2 x 1OS/well) in MEM-5% FBS.
The cultures were washed, and the cells were incubated in 0.5 ml serum-free DMEM-F
12 medium (for elastase assay) or medium containing 100 pg/ml plasminogen for 72 hr (for angiostatin activity assay). The medium was collected and centrifuged at 3000 x g at 4°C for 30 min. The supernatants were used in the assays as described below.
1 S Elastase assay Elastase activity in the conditioned medium was determined by a method described previously using [3H]NaBH4-reduced elastin as a substrate (Werb et al., 1986).
The samples were mixed in reaction buffer ( 100 mM Tris/HCI, 5 mM CaCl2, 0.2 mg/ml SDS and 0.006%
NaH3) and incubated at a concentration of 600 mg/ml at 37°C for 16 hr.
Free-form 3H release was monitored, and the enzyme activity was expressed as cpm/reaction.
Immunohistochemistry Macrophages in B 16-F 10-GM tumors were identified by using the macrophage-specific scavenger receptor antibody (Hughes et al., 1994). Sections (8-10 mm) of frozen tumor tissue or cultured cells fixed with 0.125% glutaraldehyde in PBS were treated with a rat monoclonal scavenger receptor antibody followed by gold-labeled secondary antibody as described in detail previously (Bucana et al., 1992).
Angiostatin Assay Angiostatin activity, i.e., inhibition of endothelial cell proliferation, was determined as described previously (O'Reilly et al., 1994b). Briefly, BCE were seeded onto gelatinized 24-well plates at 1.25 x 104/well/0.5 ml DMEM-10% CS, allowed to adhere overnight, rinsed, and incubated with 0.25 ml/well DMEM-5% CS or test: samples for 20 min. Additional medium containing bFGF was then added to a final concentration of 1 ng/ml at 0.5 ml/well. Seventy-two hours later, the cells were harvested by trypsinization and counted.
Angiostatin activity was expressed as percent inhibition of endothelial cell growth in culture.
RNA Isolation and Northern Blot Analyses The mRNA was extracted from tumor tissue or cell cultures using the Fast-Track kit (Invitrogen, San Diego, CA). One ~.g mRI~i~A was fractionated in 1 %
denaturing formaldehyde/agarose gels, electrotransferred to GeneScreen nylon membrane (DuPont Co., Boston, MA), and UV cross-linked with 120,000 mJ/cm2 using a UV Stratalinker (Stratagene, La Jolla, CA). Hybridization was performed as previously described (Dong et al., 1994). The filters were washed at SO-60°C with 30 mM NaCI/3 mM sodium citrate, pH 7.2/0.1 sodium dodecyl sulfate. The probes used were; cDNA fragments corresponding to rat glyceraldehyde-3-phosphate dehydrogenase (GAPD:H) , GM-CSF, and MME (Shapiro et al., 1992).
Western Blot Analysis Samples isolated from culture supernatants were mixed with sample buffer (62.5 mM
Tris/HCI, pH 6.8, 2.3% SDS, 100 mM DTT, and 0.05% bromophenol blue), boiled, and separated on 10% SDS-PAGE. The protein was transferred onto 0.45 mm nitrocellulose membranes. The filter was blocked with 3% BSA in TBS (20 mM Tris/HCI, pH 7.5, 150 mM
NaCI), probed with antibody against the lysine binding site I ( 1 ~g/ml) in TTB S (TB S containing 0.1 % Tween 20), incubated with a second antibody in the buffer, and visualized by the ECL
Western blotting detection system (bong et al., 1993a).
II. Expression of GM-CSF in engineered B16-F10 melanoma and K1735M2 melanoma cells B 16-F 10-GMCSF cells were subcloned in tissue culture to derive B 16-F 10-GMCSF(control), B 16-F 10-GMCSF(medium), and B 16-F 10-GMCSF(high) lines. The F 10-GMCSF-P (the mixture of the GM-CSF gene-transduced B 16-F 10), B 16-F 10-GMCSF(control), B 16-F 10-GMCSF(medium), and B 16-F 10-GM(high) cells produced 30, < 0.01, 18, and 75 ng/106 cells/24 hr of marine GM-CSF, respectively.
Similarly, K1735-M2-GMCSF cells were subcloned in tissue culture to obtain K1735M2-GMCSF(control), GMCSF(medium), and K1735M2-GMCSF(high) cells that constitutively released <
0.01, 0.35, 1.8 ng/106 cells/24 hr of marine GM-CSF, respectively. GM-CSF was not detected in the culture supernatants of wild-type B 16-F 10, K 173 SM2, and K 173 SM2-Neo (transfected by control vector) cells.
Correlated with expression levels of GM-CSF protein, significant amount of GM-CSF
mRNA was found in the tumor cells containing the transgene of GM-CSF. Southern blot analysis indicated that the cells engineered with GM-CSF, including B 16-F 10-GMCSF-P, B 16-F 10-GMCSF(control), B 16-F 10-GMCSF(medium), and B 16-F 10-GM(high), and K

GMCSF(control), K1735M2-GMCSF(medium), and K1735M2-GMCSF(high), contained similar level of GM-CSF cDNA.
III. Effects of the tumor cells secreting GM-CSF on secretion of elastase and generation of angiostatin by macrophages Tumor cells of B 16-F 10 lines (FIG. 12A and FIG. I2B) or K1735M2 lines (FIG.
13A and FIG. 13B) were cultured with purified peritoneal macrophages in the absence (FIG. 12A and FIG. 13A) or presence (FIG. 12B and FIG. 13B) of 100 ~g/ml of human plasminogen. The inventors found that macrophages incubated with tumor cells constitutively releasing GM-CSF, but not wild-type or non-GM-CSF releasing cells, secreted significantly higher amounts of elastase activity (FIG. 12A and FIG. 13A). In the parallel cultures containing plasminogen, significant amounts of angiostatin were found by the western blot analysis and the bioassay measuring suppression of the growth of bovine capillary endothelial cells (FIG. 12B and FIG. 13B).
IV. Macrophage infiltration in the subcutaneous tumors and expression of GM-CSF
and MME
Immunohistochemistry staining of cryostat sections using marine macrophage-specific antibody scavenger receptor revealed that B 16-F 10-GMCSF(high) tumors contained significant more infiltrating macrophages than those of B 16-F 10-GMCSF(medium) and B 16-F

GMCSF(control) tumors.

The inventors next examined the expression of GM-CSF and MME in the tumors.
GM-CSF mRNA was detected in B 16-F 10-GMCSF(high), B 16-F 10-GMCSF(medium) and F 10-GMCSF-P, but not in B 16-F 10 or B 16-F 10-GMCSF(control) tumors.
Moreover, correlated with extent of macrophage infiltration, the B 1 G-F 1 OGMCSF(high) tumors expressed a significantly higher level of GM-CSF mRNA than i:hat in B 16-F 10-GMCSF(medium) tumors.
Low level of MME mRNA was found in B 16-F 10 .and B 16-F 10-GMCSF(control) tumors. In contrast, B 16-F 10-GMCSF(high) and B 16-F 10-GMCSF(medium) tumors expressed significantly higher levels of MME mRNA.
V. Effects of B16-F10-GM tumors on the growth of distant tumors in nude mice Three models were used to determine whether tumors engineered to constitutively release GM-CSF could modulate the growth of a distant tumor. To exclude the involvement of T-cells in these process, which may complicate interpretation of the results, nude mice were used in these studies. In the first model, the inventors inve;~tigated whether B 16-F
10-GMCSF tumors could suppress the growth of lung metastasis of 3LL-met cells. B 16-F 10-GMCSF(high) or B 16-F 10-GMCSF(control) cells were implanted subcutaneously into nude mice. When the tumors reached 10-12 mm in diameter, control or the tumor-bearing mice received 3LL-met cells intravenously. As shown in Table 11, subcutaneous tumors of B 16-F 10-GMCSF(high), but not B16-F10-GMCSF(control), cells produced significant suppression on the growth of 3LL-met lung metastases. Similar results were observed in a second model in which K1735M2 melanoma cells were injected intravenously (Table 12). The inhibitory effect of subcutaneous B 16-F 10-GMCSF(high) tumors on the growth of lung metastases of K1735M2 cells could be abolished by removal of the subcutaneous tumors (Table 12). The suppression was not observed in mice bearing B 16-F 10-GMCSF(control) tumors (Table 11 and 12). These results, therefore, concluded that the suppression of the lung metastases was dependent on expression of GM-CSF
in the subcutaneous tumor lesions.

WO 98/22605 PCT/US97l21475 Suppression of 3LL-met lung metastasis in nude mice by GM-CSF gene-transduced B16-F10 cells ~rowin~ subcutaneous) Subcutaneous Lung metastases Lung Weight tumor median range (mg) no tumor 56 49 - 62 1074 ~ 237 B16-F10-GMCSF (high) 19* 8 - 31 356* ~ 59 B16-F10-GMCSF (control) 41 341 - 60 790 t 143 *, p < 0.05 Suppression of K1735M2 metastasis in nude mice by GM-CSF gene-transduced B16-F10 cells ~rowin~ subcutaneous) Subcutaneous tumor Lung metastases Lung Weight tumor resection median range (mg) no tumor --- 162 124 - 179 568 ~ 74 B 16-F 10-GMCSF (high) Sham surgery53 * 41 - 75 280* ~ 69 Resection 103 93 - 173 527 ~ 122 B 16-F 10-GMCSF (control) Sham surgery158 128 - 184 450 ~ 85 Resection 162 134 - 179 503 ~ 126 *, p < 0.05 In the third model, the inventors determined whether the suppression was only restricted to the growth of lung metastasis, the inventors investigated effects of the subcutaneous BI6-F10-GMCSF tumors on the growth of RENCA cells in the kidney of nude mice. The data shown in Table 13 indicated that B 16-F 10-GMCSF(high) but not B 16-F 10-GMCSF(control) tumors did slow down the growth of RENCA tumors in the kidney.

TABLE ~3 Suppression of the growth of RENCA calls in the kidney of nude mice by GM-CSF gene-transduced B16-F10 cells growing subcutaneously Subcutaneous tumor Kidney weight (mg) no tumor 439 t 141 B 16-F 10-GMCSF (control) 363 ~ 19 B16-F10-GMCSF (high) 244* t 60 *, p < 0.05 The inventors have thus successfully utilized animal models well accepted in the field to show that in three tumor systems, GM-CSF released from a subcutaneous tumor recruits macrophages into the tumor lesion and stimulates MME expression in these tumor infiltrating macrophages. MME in turn degrades plasminogen to angiostatin that circulates into distant capillary beds where it suppresses angiogenesis and hence the growth of the distant metastases.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.
While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention.
More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein whrile the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept oi° the invention as defined by the appended claims.

REFERENCES
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
Adams and Hamilton, Inflammation: Basic Principles and Clinical Correlates, J.
I. Gallin, I. M.
Goldstein, and R. Snyderman, eds., Raven Press, New York, 471, 1988.
Albini, Melchiori, Santi, Liotta, Brown, Stetler-Stevenson, J. Natl. Cancer Inst., 83:775, 1991.
Alt, Kellems, Bertino and Schimke, J. Biol. Chem., 253:1357, 1978.
Anderson, Science 226:401, 1984.
Angel, Bauman, Stein, Dellus, Rahmsdorf, and Herrlich, Mol. Cell. Biol., 7:2256, 1987a.
Angel, Baumann, Stein, Delois, Rahmsdorf, Herrlich, Mol. Cell Biol., 7:2256, 1987.
Angel, Imagawa, Chiu, Stein, Imbra, Rahmsdorf, Jonat, Herrlich, and Karin, Cell, 49:729, 1987b.
1 S Atchinson and Perry, Cell, 46:253, 1986.
Atchinson and Perry, Cell, 48:121, 1987.
Aukerman, Price, Fidler, J. Natl. Cancer Inst., 77(4):915-924, 1986.
Austyn and Gordon, Eur. J. Immunol, 11:805-815, 1981.
Bahnemann et al. Abs. Pap. ACS, 180:5. 1980.
Baichwal and Sugden, In: Kucherlapati R, ed. Gene transfer. New York: Plenum Press, pp.
117-148, 1986.
Banda and Werb, Biochem. J., 193:589-605, 1981.
Banda, Clark, Werb, J. Exp. Med., 152:1563-1570, 1980.
Banda, Ciark, Werb, J. Exp. Med., 157:1184-1196, 1983.
Banda, Werb, McKerrow, Methods Enzymol., 144:288, 1987.
Banerji, Olson, and Schaffner, Cell, 35:729, 1983.
Banerji, Rusconi, and Schaffner, Cell, 27:299, 1981.
Bao, Zhang, Kuo, Hum. Gene Ther., 7:355-365,1996.

Bar-Schavit, Teitelbaum, Sticklin, Eisen, Kahn, Welgus, Proc. Natl. Acad. Sci.
USA, 82:5380, 1985.
Barrett, Methods Enzymol., 244:1, 1994.
Barrett, Proteinases in Mammalian Cells and T~'ssues, A. J. Barrett, ed., North-Holland S Publishing Co., Amsterdam, 1, 1977.
Bass, Cabrera, Elgavish, Robert, Siegal, Anderson, Maneval, Curiel, Cancer Gene Ther., 2:97-I04, 1995.
Basset, Bellocq, Wolf, Stoll, Limacher, Podhajeer, Chenard, Rio, Chambon, Nature, 348:699, 1990.
Belaaouaj, Shipley, Kobayashi, Zimonjic, Popescu, Silverman, Shapiro, J. Biol.
Chem., 270:14568-14575, 1995.
Belardelli et al., J. Gen. Virol., 68:2203-2212,1987a.
Belardelli, Vignaux, Proietti, Gresser, Proc. Natl. Acad. Sci. USA, 81:602-606,1987b.
Berkhout, Silverman, and Jeang, Cell, 59:273, 1989.
1 S Berzal-Herranz et al., Genes and Devel., 6:129-134, 1992.
Birkeal-Hansen, Moore, Bodden, Windsor, Birkedal-Hansen, DeCarlo, Engler, Crit. Rev. Oral Biol. Med., 4:197, 1993.
Blake, Allen, Lunec, Br. Med. Bull., 43:371, 1987.
Blanar, Baldwin, Flavell, and Sharp, EMBO J., 8:1139, 1989.
Bodine and Ley, EMBO J., 6:2997, 1987.
Boshart, Weber, Jahn, Dorsch-Hasler, Fleckenstein, and Schaffner, Cell, 41:521, 1985.
Bosze, Thiesen, and Charnay, EMBO J., 5:1615, 1986.
Brach and Hernnan, Acta Haematol., 86:128, 1991.
Braddock, Chambers, Wilson, Esnouf, Adams, Kingslnan, and Kingsman, Cell, 58:269, 1989.
Brody, Metzger, Danel, Rosenfeld, Crystal, Hum. Gene Ther., 5:821-836,1994.
Bucana, Fabra, Sanchez, Fidler, Am. J. Pathol., 141:1;?25-1236, 1992.
Bulla and Siddiqui, J. Virol., 62:1437, 1986.
Busiek, Ross, McDonnell, Murphy, Matrisian, Welgus, J. Biol. Chem., 267:9087-9092, 1992.
Campbell, Laboratory Techniques in Biochemistry and Molecular Biology, Vol.
I3, Burden and Von Knippenberg, Eds. pp. 75-83, Amsterdam, Elseview, 1984.

Campbell and Villarreal, Mol. Cell. Biol., 8:1993, 1988.
Campbell, Cury, Lazarus, Welgus, J. Biol. Chem., 262:15862, 1987.
Campbell, Cury, Shapiro, Goldberg, and Welgus. J. Immunol. 146:1286, 1991.
Campere and Tilghman, Genes and Dev., 3:537, 1989.
Campo, Spandidos, Lang, and Wilkie, Nature, 303:77, 1983.
Capaldi et al., Biochem. Biophys. Res. Comm., 76:425, 1977.
Cech et al., Cell, 27:487-496, 1981.
Celander and Haseltine, J. Virology, 61:269, 1987.
Celander, Hsu, and Haseltine, J. Virology, 62:1314, 1988.
Chalfie et al., Science, 263:802-805,1994.
Chandler, Maler, and Yamamoto, Cell, 33:489, 1983.
Chang et al., Hepatology, 14:134A, 1991.
Chang, Barr, Seltzer, Jiang, Nabel, Nabel, Parmacek, Leiden, Science, 267:518-525,1995.
Chang, Erwin, and Lee, Mol. Cell. Biol., 9:2153, 1989.
1 S Chatterjee, Lee, Rentoumis, and Jameson, Proc. Natl. Acad. Sci. U.S.A., 86:9114, 1989.
Cheeseman and Slater. Br. Med. Bull., 49:481, 1993.
Chen, Clark, Chon. Blood, 71:997, 1988.
Choi, Chen, Kriegler, and Roninson, Cell, 53:519, 1988.
Chowrira et al., Biochemistry, 32:1088-1095, 1993.
Chowrira et al., J.B.C., 269:25856-25864, 1994.
Chu, Sperber, Mayer, Hsu, Virology, 188:793-800,1992.
Clark, Voulgaropoulou, Fraley, and Johnson, Human Gene Therapy, 6:1329-1341, 1995.
Coffin, Virology, Fields et al. (eds.), New York: Raven Press, pp. 1437-1500, 1990.
Cohen, Walter, and Levinson, J. Cell. Physiol., 5:75, 1987.
Colberre-Garapin et al., .I. Mol. Biol., 150:1, 1981.
Costa, Lai, Grayson, and Darnell, Mol. Cell. Biol., 8:81, 1988.

Cotten, Wagner, Zatloukal, Phillips, and Curiel, P. N .A. S. USA, 89:6094-6098, 1992.
Couch et al., Am. Rev. Resp. Dis., 88:394-403, 1963.
Coupar et al., Gene, 68:1-10, 1988.
Cripe, Haugen, Turk, Tabatabai, Schmid, Durst, i3issmann, Roman, and Turek, EMBO J., 6:3745, 1987.
Croen,J. Clin. Invest., 91:2446-2452,1993.
Crosier and Clark, Semin. Oncol., 19:349, 1992.
Crystal et al., Nat. Genet., 8:42-50,1994.
Crystal, Science, 270:404-410,1995.
Culotta and Hamer, Mol. Cell. Biol., 9:1376, 1989.
Curiel, Viruses in Human Gene Therapy, J.-M.H. Vos (Ed.), Carolina Academic Press, Durham, N.C., pp. 179-212, 1994.
Cury, Campbell, Lazarus,.Albin, Welgus, J. Immunol., 141:4306, 1988.
Dandolo, Blangy, and Kamen, J. Virology, 47:55, 1983.
I5 De Villiers, Schaffner, Tyndall, Lupton, and Kamen, Nature, 312:242, 1984.
Dean, Martel-Pelleier, Pelleter, Howell, Woessner, Jr.., J. Clin. Invest., 84:678, 1989.
DeClerck, Yean, Chan, Shimada, Langley, Cancer RE~s., 51:2151, 1991.
Deisseroth et al., Blood, 83:3068-3076,1994.
Deisseroth,Hum. Gene Ther., 4:71-85, 1993.
Deschamps, Meijlink, and Verma, Science, 230:1174., 1985.
Devlin, Devlin, Myambo, Lilly, Rado, Warren, J. Leukoc. Biol., 41:302, 1987.
Ding, Nathan, Stuehr, J. Immunol., 141:2407, 1988.
Dinney, Bucana, Utsugi, Fidler, von Eschenbach, Killion, Cancer Res, 51:3741-3747, 1991.
Docherty, Lyons, Smith, Wright, Stephens, Harris, Murphy, Reynolds, Nature 318:66, 1985.
Dong, Qi, Fidler, J. Exp. Med., 173:1071-1077, 1993x.
Dong, Qi, Xie, Fidler, J. Immunol, 151:2717-2724, 1993b.
Dong, Radinsky, Fan, Tsan, Bucana, Wilmanns, Fidler, J. Natl. Cancer Inst., 86:913-920, 1994x.

Dong, Staroselsky, Qi, Xie, Fidler, Cancer Res., 54:789-793,1994b.
Dong, Wang, Van Ginkel, Pascual, Frizzell, Hum. Gene Ther., 7:319-331,1996.
Dong, Yang, Xie, Juang, Llansa, Fidler, J. Leukoc. Biol., 58:725-732, 1995.
Dranoff, Jaffee, Lazenby, Golumbek, Levitsky, Brose, Jackson, Hamada, Pardoll, Mulligan, Proc. Natl. Acad. Sci. USA, 90:3539-3543, 1993.
Dudon and Vogel, Infect. Immunol., 49:709, 1985.
Edbrooke, Burt, Cheshire, and Woo, Mol. Cell. Biol., 9:1908, 1989.
Edlund, Walker, Barr, and Rutter, Science, 230:912, 1985.
Fang, Eisensmith, Wang, Kay, Cross, Landen, Gordon, Bellinger, Read, Hu, Brinkhous, Woo, Hum. Gene Ther., 6:1039-1044,1995.
Feinberg and Vogelstein, Anal. Biochem., 132:6, 1983.
Feng and Holland, Nature, 334:6178, 1988.
Fidler, Biomedicine 32:1-3, 1980.
Fidler, Cancer Res., 50:6130-6138, 1990.
Fidler, Cancer Res., 45:4714-4726, 1985.
Fidler, J. Natl. Cancer Inst., 87:1588, 1995.
Fidler, Res. Immunol., 143:199-204, 1992.
Fidler, Adv. Pharmacol., 30:271-326, 1994.
Fidler and Balch, Curr. Probl. Surg. 24:137-208, 1987.
Fidler and Ellis, Cell, 79:185-188, 1994.
Fidler and Poste, Semin. Oncol. 12:207-221, 1985.
Fidler and Schroit, Biochim. Biophys. Actor, 948:151-173, 1988.
Firak and Subramanian, Mol. Cell. Biol., 6:3667, 1986.
Flotte, Afione, Conrad, McGrath, Solow, Oka, Zeitlin, Guggino, and Carter, Proc. Natl. Acad Sci. USA, 90:10613-10617, 1993.
Flotte, Barraza-Ortiz, Solow, Afione, Carter, and Guggino, Gene Therapy, 2:29-37, 1995.
Flotte, Solow, Owens, Afione, Zeitlin, and Carter, Am. J. Respir. Cell Mol.
Biol., 7:349-356, 1992.

Foecking and Hofstetter, Gene, 45:101, 1986.
Folkman, Cancer Res. 46, 467-473, 1986.
Folkman, Nature Med., 1:27-31,1995.
Folkmanand Shing,J. Biol. Chem., 267:10931-10934" 1992.
Forster and Symons, Cell, 49:211-220, 1987.
Fort, Morty, Piechaczyk, Sabrouty, Dani, Jeanteur, Blanchard, Nucleic Acids Res., 13:1431, 1985.
Foulke, Marshall, Trotta, Von Hoff, Cancer Res., 50:6264, 1990.
Fraser, Hughes, Gordon, Nature, 364:343-346, 1993.
Friedmann, Science, 244:1275-1281) 1989.
Fu, Watson, Jimenez, Wang, Lopez, Cancer Res., 50:227, 1990.
Fujihara, Ito, Pace, Watanabe, Russell, Suzuki, J. Biol. Chem., 269:12?73-12778,1994.
Fujita, Shibuya, Hotta, Yamanishi, and Taniguchi, Cell, 49:357, 1987.
Gefter et al., Somatic Cell Genet. 3:231-236, 1977.
Gerlach et al., Nature (London), 328:802-805, 1987.
Gessani, Belardelli, Borghi, Boraschi, Gresser,J. Immimol., 139:1991-1998,1987.
Ghosh-Choudhury et al., EMBO J., 6:1733-1739, 198.7.
Ghosh and Bachhawat, Liver diseases, targeted diagnosis and therapy using specific receptors and ligands, Wu G. and C. Wu ed. New York: Marcel Dekker, pp. 87-104, 1991.
Gilles, Morns, Oi, and Tonegawa, Cell, 33:717, 1983.
Ginsberg et al., Proc. Natl. Acad. Sci. USA, 88:1651-1655,1991.
Gius, Grossman, Bedell, and Laimins, J. Virology, 62:665, 1988.
Gloss, Bernard, Seedorf, and Klock, EMBO J., 6:373.'i, 1987.
Godbout, Ingram, and Tilghman, Mol. Cell. Biol., 8:1169, 1988.
Goding, Monoclonal Antibodies: Principles and Practice, 2d ed., Orlando, Fla., Academic Press, 1986, pp. 60-61, 65-66, 71-74.
Gornez-Foix et al., J. Biol. Chem., 267:25129-25134, 1992.
Goodbourn and Maniatis, Proc. Natl. Acad Sci. USA, 85:1447, 1988.

Goodbourn, Burstein, and Maniatis, Cell, 45:601, 1986.
Gorelik, Adv. Cancer Res., 39:71-120, 1983a.
Gorelik, Cancer Res., 43:138-145, 1983b.
Gorelik, Segal, Feldman, Int. J. Cancer, 27:847-856, 1981.
Graham et al., J. Gen. Virol., 36:59-72, 1977.
Graham and PreveY, Gene Transfer and Expression Protocols, Murray, E.J., ed., Humana, New Jersey, vol. 7, 109-128,1991.
Graham and Prevec, Biotechnology, 20:363-390, 1992.
Greene, Bohnlein, and Ballard, "Immunology Today, 10:272, 1989.
Grosschedl and Baltimore, Cell, 41:885, 1985.
Grubb et al., Nature, 371:802-806,1994.
Grunhaus and Horwitz, Seminar in Virology, 3:237-252, 1992.
Gutterman,Proc. Natl. Acad. Sci. USA, 91:1198-1205,1994.
Guzman, Hirschowitz, Brody, Crystal, Epstein, Finkel, Proc. Natl. Acad. Sci.
USA, 91:10732-10736,1994.
Hanania and Deisseroth, Cancer Gene Ther., 1:21-25, 1994.
Hart and Fidler, Biochem. Biophys. Acta, 651:37-50,1981.
Haseloff and Gerlach, Nature, 334:585-591, 1988.
Haslinger and Karin, Proc. Natl. Acad. Sci. U.S.A., 82:8572, 1985.
Hauber and Cullen, J. Virology, 62:673, 1988.
Hen, Borrelli, Fromental, Sassone-Corsi, and Chambon, Nature, 321:249, 1986.
Henney, Wakely, Davies, Foster, Henbry, Murphy, Humphries, Proc. Natl. Acad.
Sci. USA, 88:815, 1991.
Hensel, Meichle, Pfizenmaier, and Kronke, Lymphokine Res., 8:347, 1989.
Hermonat and Muzyczka, Proc. Nat'l. Acad. Sci. USA, 81:6466-6470, 1984.
Herr and Clarke, Cell, 45:461, 1986.
Hersdorffer et al., DNA Cell Biol., 9:713-723, 1990.
Herz and Gerard, Proc. Nat'1 Acad. Sci. USA 90:2812-2816, 1993.

Hibbs, Hoidal, Kang, J. Clin. Invest., 80:1644, 1987.
Hibbs, Taintor, Vavrin, Science, 235:473, 1987.
Higashi, Sokawa, Watanabe, Kawade, Ohno, Takaoka, Taniguchi, J. Biol. Chem., 258:9522-9529, 1983.
Hirochika, Browker, and Chow, J. Virol., 61:2599, I S>87.
Hirsch, Gaugler, Deagostini-Bauzin, Bally-Cuif, and Gordis, Mol. Cell. Biol., 10:1959, 1990.
Holbrook , Gulino, and Ruscetti, Yirolo~, 157:211, 1987.
Holmgren L, O'Reilly MS, Folkman J., Nature Med 1995;149.
Horlick and Benfield, Mol. Cell. Biol., 9:2396, 1989.
Horwich et al., J. Virol., 64:642-650, 1990.
Huang, Ostrowski, Berard, and Hagar, Cell, 27:245, 1981.
Hug, Costas, Staeheli, Aebi, and Weissmann, Mol. CE~II. Biol., 8:3065, 1988.
Hughes, Fraser, Gordon, Immunology Letters, 43:7-1 ~l, 1994.
Hwang, Lim, and Chae, Mol. Cell. Biol., 10:585, 1990.
Imagawa, Chiu, and Karin, Cell, 51:251, 1987.
Imbra and Karin, Nature, 323:555, 1986.
Imler, Lemaire, Wasvlyk, and Waslyk, Mol. Cell. Biol., 7:2558, 1987.
Imperiale and Nevins, Mol. Cell. Biol., 4:875, 1984.
Ioannides, Freedman, Platsoucas et al., J. Immunol., 146:1700-1705,1991.
Jakobovits, Smith, Jakobovits, and Capon, Mol. Cell. Biol., 8:2555, 1988.
Jameel and Siddiqui, Mol. Cell. Biol., 6:710, 1986.
Jaynes, Johnson, Buskin, Gartside, and Hauschka, Mol. Cell. Biol., 8:62, 1988.
Johnson, Wold, and Hauschka, Mol. Cell. Biol., 9:3393, 1989a.
Jones and Castro, Br. J. Cancer, 35:519-527, 1977.
Jones and Shenk, Cell, 13:181-188, 1978.
Joraschkewitz, Depenbrock, Freund, Erdmann, Meyer, DeRiese, Nenkem, Hanauske, Krumwieh, Poliwade, Hanaciske, Eur. J. Cancer, 26:1070, 1992.

Joyce, Nature, 338:217-244, 1989.
Kadesch and Berg, Mol. Cell. Biol., 6:2593, 1986.
Kaneda et al., Science, 243:375-378, 1989.
Kaplitt, Leone, Samulski, Siao, Pfaff, O'Malley, and During, Nature Genetics, 8:148-154, 1994.
Karin, Haslinger, Heguy, Dietlin, and Cooke, Mol. Cell. Biol. , 7:606, 1987.
Karlsson et al., EMBO J., 5:2377-2385, 1986.
Karupiah, Xie, Butler, Nathan, Duarte, MacMicking, Science, 261:1445-1448,1993.
Katinka, Vasseur, Montreau, Yaniv, and Blangy, Nature, 290:720, 1981.
Katinka, Yaniv, Vasseur, and Blangy, Cell, 20:393, 1980.
Kato et al., J. Biol. Chem., 266:3361-3364, 1991.
Katsuda, Okada, Okada, Imai, Nakanishi, Am. J. Pathol., 145:1208-1218, 1994.
Kaufman, Methods in Enrymology, 185:537-566, 1990.
Kawamoto, Makino, Niw, Sugiyama, Kimura, Anemura, Nakata, and Kakunaga, Mol.
Cell. Biol., 8:267, 1988.
Kay, Landen, Rothenberg, Taylor, Leland, Wiehle, Fang, Bellinger, Finegold, Thompson, Read, Brinkhous, Woo, Proc. Natl. Acad. Sci. USA, 91:2353-2357,1994.
Kelleher and Vos, Biotechniques, 17(6):1110-1117, 1994.
Keller, Adv. Exp. Med. Biol., 155:289-302, 1982.
Kerr, Miller, Matrisian, Cell, 61:267, 1990.
Khokha, Waterhouse, Yagel, Lale, Overall, Norton, Denhardt, Science, 243:947, 1989.
Kiledjian, Su, and Kadesch, Mol. Cell. Biol., 8:145, 1988.
Kim and Cech, Proc. Natl. Acad. Sci. USA, 84:8788-8792, 1987.
Klamut, Gangopadyhay, Worton, and Ray, Mol. Cell. Biol. , 10:193, 1990.
Klein et al., Nature, 327:70-73, 1987.
?S Knight, Proteinases in Mammalian Cells and Tissues, A. J. Barrett, ed., North-Holland Publishing, Co., Amsterdam, 583, 1977.
Knowles, Hohneker, Zhou, Olsen, Noah, Hu, Leigh, Engelhardt, Edwards, Jones, Grossman, Wilson, Johnson, Boucher, N. Engl. J. Med., 333:823-831,1995.

Koch, Benoist, and Mathis, Mol. Cell. Biol., 9:303, 1989.
Kohler and Milstein, Nature, 256:495-497, 1975.
Kohler and Milstein, Eur. J. Immunol., 6:511-519, 19'76.
Kohn, Hollister, DiPersio, Wahl, Liotta, Schiffman, Int. J. Cancer, 53:968, 1993.
Kotin, Siniscalco, Samulski, Zhu, Hunter, McLaughlin, Muzyczka, and Berns, Proc. Natl.
Acad.,Sci. USA, $7:2211-2215, 1990.
Kriegler and Botchan, Eukaryotic Viral Vectors, ed. Y. Gluzman. Cold Spring Harbor: Cold Spring Harbor Laboratory, NY, 1982.
Kriegler and Botchan, Mol. Cell. Biol. 3:325, 1983.
Kriegler, Perez, and Botchan, Gene Expression, eds. D. Hamer and M. Rosenberg.
New York:
Alan R. Liss, 1983.
Kriegler, Perez, Defay, Albert and Liu, Cell, 53:45, 1 ~~88.
Kriegler, Perez, Hardy and Botchan, Cell, 38:483, i 984a.
Kriegler, Perez, Hardy, and Botchan, Cancer Cells 2,iOncogenes and Viral Genes, G. F. Van de Woude, A. J. Levine, W. C. Topp, and J. D. Watson, Cold Spring Harbor: Cold Spring Harbor Laboratory, 1984b.
Kuhl, De La Fuenta, Chaturvedi, Parinool, Ryals, Meyer, and Weissman, Cell, 50:1057, 1987.
Kumar, Dong, Fidler, J. Immunol., 1996.
Kunz, Zimmerman, Heisig, and Heinrich, Nucl. Acids Res., 17:1121, 1989.
Lacraz, Nicod, Rochemonteix, Baumberger, Dayer, Welgus, J. Clin. Invest., 90:382-388, 1992.
LaFace, Hermonat, Wakeland, and Peck, Viology, 162:483-486, 1988.
Lallemand, Lebon, Blanchard, Tovey, J. Leukoc. Biol., 60:137-146,1996.
Larsen, Harney, and Moore, Proc. Natl. Acad. Sci. U.~S.A., 83:8283, 1986.
Larsson and Litwin, Dev. Biol. Standard., 66:385-390, 1987.
Laspia, Rice, and Mathews, Cell, 59:283, 1989.
Latimer, Bergen and Baumann, "Highly Conserved Upstream Regions of the a~-Antitrypsin Gene in Two Mouse Species Govern Liver-Specific Expression by Different Mechanisms," Mol. Cell. Biol., 10:760, 1990.
Laughlin, Cardellichio, and Coon, J. Virol., 60:515-5:?4, 1986.
Le Gal La Salle et al., Science, 259:988-990, 1993.

Lebkowski, McNally, Okarma, and Lerch, Mol. Cell. Biol., *:3988-3996, 1988.
Lee, Mulligan, Berg, and Ringold, Nature, 294:228, 1981.
Levinson, Khoury, VanDeWoude, and Gruss, Nature, 295:79, 1982.
Levrero et al., Gene, 101:195-202, 1991.
Levy, Cioce, Sobel, Garbisa, Grigioni, Liotta, Stetler-Stevenson, Cancer Res., 5 i ( 1 ):439-444, 1991.
Lieber and Strauss, Mol. Cell. Biol., 15: 540-551, 1995.
Lim, US Patent 4,352,883, October 5, 1982.
Lin, Cross, Halden, Dragos, Toledano, and Leonard, Mol. Cell. Biol., 10:850, 1990.
Luo, Zhou, Cooper, Munshi, Boswell, Broxmeyer, and Srivastava, Blood,82 (Supp.): 1,303A, 1994.
Luria, Gross, Horowitz, and Givol, EMBO J., 6:3307, 1987.
Lusky and Botchan, Proc. Natl. Acad. Sci. U.S.A., 83:3609, 1986.
Lusky, Berg, Weiher, and Botchan, Mol. Cell. Biol. 3:1108, 1983.
MacGregor, Nolan, Fiering, Roederer, Herzenber, Gene Transfer and Expression Protocols, Murray, E.J., ed., Humans, New Jersey, vol. 7, 217-235,1991.
Majors and Varmus, Proc. Natl. Acad. Sci. U.S.A., 80:5866, 1983.
Mann et al., Cell, 33:153-159, 1983.
Mantovani, Bottazzi, Colotta, Sozzani, Ruco, Immunol. Today, 13:265-270, 1992.
Mantovani, Giavazzi, Polentarutti, Spreafico, Garattini, Int. J. Cancer, 25:617-620, 1980.
Markowitz et al., J. Virol., 62:1120-1124, 1988.
Marshall, Science, 269:1050-1055,1995.
Matrisian, Trends Genet., 6:121, 1990.
Matrisian, BioEssays, 14:455-463, 1992.
McCarty, Christensen, and Muzyczka, J. Virol. , 65:2936-2945, 1991.
McLaughlin, Collis, Hermonat, and Muzyczka, J. Virol., 62:1963-1973, 1988.
McNeall, Sanchez, Gray, Chesterman, and Sleigh, Gene, 76:81, 1989.
Melkova and Esteban, J. Immunol., 155:5711-5718,1995.

Metcalf, Cancer, 65:2185, 1990.
Michel and Westhof, J. Mol. Biol., 216:585-610, 1990.
Mignatti, Robbins, Rifkin, Cell, 47:487, 1986.
Miksicek, Heber, Schmid, Danesch, Posseckert, Beato, and Schutz, Cell, 46:203, 1986.
Mizrahi, Process Biochem., (August):9-12, 1983.
Moncade, Palmer, Higgs, Pharmacol. Rev. , 43 :109, 1991.
Mordacq and Linzer, Genes and Dev., 3:760, 1989.
Moreau, Hen, Wasylyk, Everett, Gaub, and Chambon., Nucl. Acids Res., 9:6047, 1981.
Morikawa, Walker, Nakajima, Pathak, Jessup, Fidler, Cancer Res., 48:6863-6871, 1988.
Murphy and Docherty, Am. J. Resp. Cell Mol. Biol., i':120, 1992.
Muesing, Smith, and Capon, Cell, 48:691, 1987.
Muzyczka, Curr. Top. Microbiol. Immunol., 158:97-1.29, 1992.
Nakajima, Morikawa, Fabra, Bucana, Fidler, J. Natl. Cancer Inst., 82:1890, 1990.
Nakamura, Merchav, Carter, Ernst, Demetri, Fumkawa, Anderson, Freedman, Griffin, J.
Immunol., 143:3543, 1989.
Nathan, Fed. Proc., 41:2206, 1982.
Nathan, J. Clin. Invest., 79:319-326, 1987.
Nicolas and Rubinstein, Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt (eds.), Stoneham: Butterworth, pp. 494-513, 1988.
Nilsson and Mosbach, Dev. Biol. Standard , 66:183-193, 1987.
Ng, Gunning, Liu, Leavitt, and Kedes, Nuc. Acids Res., 17:601, 1989.
Normann, Cancer Metastasis., (Rev. 4}, 277-291, 19F~5.
O'Reilly, Holmgren, Chen, Folkman, Nature Med., 2:689-692, 1996.
O'Reilly, Holmgren, Shing, Chen, Rosenthal, Moses, Lane, Cao, Sage, Folkman, Cell, 79:315-~5 328, 1994.
Ohi, Dixit, Tillery, and Plonk, Gene, 89L:279-282, 1990.
Ohlsson and Olsson, Eur. J. Biochem., 42:519, 1974.
Ondek, Sheppard, and Herr, EMBO J., 6:1017, 1987.

Ornitz, Hammer, Davison, Brinster, and Palmiter, Mol. Cell. Biol., 7:3466, 1987.
Palmiter, Chen, and Brinster, Cell, 29:701, 1982.
Palukaitis et al., Virology, 99:145-151, 1979.
Paskind et al., Virology, 67:242-248, 1975.
Pech, Rao, Robbins, and Aaronson, Mol. Cell. Biol., 9:396, 1989.
Perez-Stable and Constantini, Mol. Cell. Biol., 10:1116, 1990.
Perriman et al., Gene, 113:157-163, 1992.
Petricciani, Dev. Biol. Standard., 66:3-12, 1985.
Pieard and Schaffner, Nature, 307:83, 1984.
Phillips et al., Large Scale Mammalian Cell Culture (Feder, J. and Tolbert, W.
R., eds.), Academic Press, Orlando, FL, U.S.A., 1985.
Pinkert, Ornitz, Brinster, and Palmiter, Genes and Dev., 1:268, 1987.
Polverini, Macrophage-derived Cell Regulatory Factors, C. Sorg, ed., (Basel:
Karger), 54-73, 1989.
Ponta, Kennedy, Skroch, Hynes, and Groner, Proc. Natl. Acad. Sci. U.S.A., 82:1020, 1985.
Porton, Zaller, Lieberson, and Eckhardt, Mol. Cell. Biol., 10:1076, 1990.
Poste and Fidler, Nature, 283:139-146,1980.
Prehn, Cancer Res., S 1:2-4, 1991.
Prehn, Cancer Res., 53:3266-3269, 1993.
Prody et al., Science, 231, 1577-1580, 1986.
Queen and Baltimore, Cell, 35:741, 1983.
Quinn, Farina, Gardner, Krutzsch, and Levens, Mol. Cell. Biol., 9:4713, 1989.
Racher et al., Biotechnology Techniques, 9:169-174, 1995.
Radinsky, Kraemer, Raines, Xung, Culp, Proc. Natl. Acad Sci. USA, 84:5143, 1987.
Ragot et al., Nature, 361:647-650, 1993.
Ramakrishnan, Brandt, Niedel, Bast, Jr., Brown, J. Clin. Invest., 83:921, 1989.
Redondo, Hata, Brocklehurst, and Krangel, Science, 247:1225, 1990.

Reinhold-Hurek and Shub, Nature, 357:173-176, 1992.
Reisman and Rotten Mol. Cell. Biol., 9:3571, 1989.
Renan, Radiother. Oncol., 19:197-218, 1990.
Resendez Jr., Wooden, and Lee, Mol. Cell. Biol., 8:4~~79, 1988.
Rich et al., Hum. Gene Ther., 4:461-476, 1993.
Ridgeway, Rodriguez RL, Denhardt DT, ed. Vectors: A survey of molecular cloning vectors and their uses. Stoneham: Butterworth, pp. 467-492, 1988.
Ripe, Lorenzen, Brenner, and Breindl, Mol. Cell. Biol., 9:2224, 1989.
Riffling, Coutinho, Amarm, and Kolbe, Nuc. Acids Res., 17:1619, 1989.
Rosen, Sodroski, and Haseltine, Cell, 41:813, 1988.
Rosenberg, Biologic Therapy of Cancer, DeVita VT, Hellman S, Rosenberg SA, eds., 2nd ed., pp.
487-506, Philadelphia, J.B. Lippincott Co., 1995.
Rosenfeld, Siegfried, Yoshimura, Yoneyama, , Fukayama, Stier, Paakko, Gilardi, Stratford-Perricaudet, Perncaudet, Jallat, Pavirani, Lecocq, Crystal, Science, 252:431-434,1991.
Rosenfeld, Yoshimura, Trapnell, Yoneyama, Rosenthal, Dalemans, Fukayama, Bargon, Stier, Stratford-Perricaudet, Perncaudet, Guggino, Pavirani, Lecocq, Crystal, Cell, 68:143-155, 1992.
Roux et al., Proc. Natl. Acad. Sci. USA, 86:9079-9083, 1989.
Saiki and Fidler, J. Immunol., 135:684, 1985.
Sakai, Helms, Carlstedt-Duke, Gustafsson, Rottman, and Yamamoto, Genes and Dev., 2:1144, 1988.
Sampson-Johannes and Cerlino, J. Immunol., 141:3680, 1988.
Samulski, Chang, and Shenk, J. Virol., 63:3822-3828, 1989.
Samulski, Zhu, Xiao, Brook, Housman, Epstein, and Hunter, EMBOJ., 10:3941-3950, 1991.
Santerre, et al., Gene, 30:147, 1984.
Sarver et al, Science, 247:1222-1225, 1990.
Satake, Furukawa, and Ito, J. Virology, 62:970, 1988.
Scanlon et al., Proc Natl Acad Sci USA, 88:10591-10'.>95, 1991.
Schaffner, Schirm, Muller-Baden, Wever, and Schaffr~er, J. Mol. Biol., 201:81, 1988.

Schreck, Rieber, Baeuerle, EMBO J., 10:2247, 1991.
Searle, Stuart, and Palmiter, Mol. Cell. Biol., 5:1480, 1985.
Sen and Lengyel, J. Biol. Chem., 267:5117-5120,1992.
Sen and Ransohoff,Adv. Virus Res., 42:57-102,1993.
Senior, ConnoIly, Cury, Welgus, Campbell, Am. Rev. Respir. Dis., 139:1251, 1989.
Senior, Griffin, Fliszar, Shapiro, Goldberg, Welgus, J. Biol. Chem., 266:7870-7875, 1991.
Sessa, Harrison, Barber, Zeng, Durieux, D'Angelo, Lynch, Peach, J. Biol.
Chem., 267:15274, I 992.
Shapiro, Am. J. Respir. Crit. Care Med , 150:5160, 1994.
Shapiro, Campbell, Kobayashi, Welgus, J. Immunol., 146:2724, 1991.
Shapiro, Campbell, Kobayashi, Welgus, J. CI in. Invest. , 86:1204, 1990.
Shapiro, Griffin, Gilbert, Jenkins, Copeland, Welgus, Senior, Ley, J. Biol.
Chem., 267:4664-4671, 1992.
Shapiro, Kobayashi, Ley, J. Biol. Chem., 268:23824-23829, 1993a.
Shapiro, Kobayashi, Pentland, Welgus, J. Biol. Chem., 268:8170-8175, I993b.
Shapiro, Kobayashi, Welgus, J. Biol. Chem., 267:13890, 1992.
Sharp and Marciniak, Cell, 59:229, 1989.
Shaul and Ben-Levy, EMBO J., 6:1913, 1987.
Sherman, Basta, Moore, Brown, and Ting, Mol. Cell. Biol., 9:50, 1989.
Shipley, Doyle, Fliszar, Ye, Johnson, Shapiro, Welgus, Senior, J. Biol. Chem., 271:4335-4341, 1996a.
Shipley, Wesselschmidt, Kobayashi, Ley, Shapiro, Proc. Natl. Acad. Sci. USA, 93:3942-3946, 1996b.
Shotton and Hartley, Nature, 225:802, 1970.
Simon et al., Hum. Gene Ther., 4:771-780,1993.
Sioud et al.,.JMoI. Biol., 223:831-835, 1992.
Sleigh and Lockett, J. EMBO, 4:3831, 1985.
Smith and Rutledge, Natl. Cancer Inst. Monogr., 42:141-143, 1975.

Sottrup-Jensen, Claeys, Zajdel, Petersen, Magnusson, Prog. Chem. Fibrinol.
Thrombol., 3:191, 1978.
Spalholz, Yang, and Howley, Cell, 42:183, 1985.
Spandau and Lee, J. Virology, 62:427, 1988.
Spandidos and Wilkie, EMBO J , 2:1193, 1983.
Staroselsky, Pathak, Chernajovsky, Tucker, Fidler, Cancer Res, 51:6292-6298, 1991.
Stephens and Hentschel, Biochem. J. , 248:1, 1987.
Stratford-Perricaudetet al., Hum. Gene. Ther. 1:241-256,1991.
Stratford-Perricaudetand Perricaudetp, Human Gene Transfer, Eds, O. Cohen-Haguenauer and M.
Boiron, Editions John Libbey Eurotext, France:, p. 51-61, 1991.
Stratford-Perricaudet,Makeh, Perricaudet, Briand, J. C.'lin. Invest., 90:626-630,1992.
Stuart, Searle, and Palmiter, Nature, 317:828, 1985.
Stuehr and Griffith, Advances in Enrymology ana' Related Areas of Molecular Biology, A.
Meister, ed., Interscience, New York, 65, 287, 1992.
Stuehr and Nathan, J. Exp. Med , 169:1543, 1989.
Sugarbaker, Curr. Prob. Cancer 3:1-59, 1979.
Sullivan and Peterlin, Mol. Cell. Biol., 7:3315, 1987.
Swartzendruber and Lehman, J. Cell. Physiology, 85:179, 1975.
Symons, R.H., Annu. Rev. Biochem., 61:641-67I, 19!2.
Symons, R.H., NA.R., 9:6527-6537, 1981.
Takebe, Seiki, Fujisawa, Hoy, Yokota, Arai, Yoshida, and Arai, Mol. Cell.
Biol., 8:466, 1988.
Takeda, Fujii, Nitta, Sakihara, Nakayama, Rikiishi, Kumagai, Jpn. J. Cancer Res., 82:1299-1308, 1991.
Takeda, Hatakeyama, Tsuchiya, Rikhshi, Kumagai, Lnt. J. Cancer, 47:413-420, 1991.
Talmadge and Fidler, J. Natl. Cancer Inst., 69:975-980, 1982.
Talmadge, Key, Fidler, J. Immunol., 126:2245-2248, 1981.
Taniguchi, Ohno, Fujii-Kuriyama,Muramatsu, Gene, 10:11-15,1980.
Tavernier, Gheysen, Duerinck, Can Der Heyden, and Fiers, Nature, 301:634, 1983.

Taylor and Kingston, Mol. Cell. Biol. , I 0:176, 1990b.
Taylor and Kingston, Mol. Cell. Biol., 10:165, 1990a.
Taylor, Solomon, Weiner, Paucha, Bradley, and Kingston, J. Biol. Chem., 264:15160, 1989.
Temin, Gene Transfer, Kucherlapati (ed.), New York: Plenum Press, pp. 149-188, 1986.
Tests and Quigley, J. Natl. Cancer Inst., 83:740, 1991.
Thiesen, Bosze, I-Ienry, and Charnay, J. Virology, 62:614, 1988.
Thompson, J.D. et al., Nature Medicine, 1:277-278, 1995.
Top et al., J. Infect. Dis., 124:155-160, 1971.
Topalian, Biologic Therapy of Cancer, DeVita VT, Hellman S, Rosenberg SA, eds., 2nd ed., pp.
467-486, Philadelphia, J.B. Lippincott Co., 1995.
Topalian, Solomon, Rosenberg,J. Immunol., 142-3714-3720,1989.
Tratschin, Miller, Smith, and Carter, Mol. Cell. Biol., 5:32581-3260, 1985.
Tratschin, West, Sandbank, and Carter, Mol. Cell. Biol., 4:2072-2081, 1984.
Treisman, Cell, 42:889, 1985.
Tronche, Ropier, Bach, Weiss, and Yaniv, Mol. Cell. Biol., 9:4759, 1989.
Tronche, Ropier, Herbomel, Bach, Cereghini, Weiss, and Yaniv, Mol. Biol. Med., 7:173, 1990.
Trudel and Constantini, Genes and Dev., 6:954, 1987.
Tsuchiya, Igarashi, Suzuki, Kumagai, J. Immunol., 141:699, 1988.
Tudor, Groves, Badesch, Voelkel,Am. J. Pathol., 144:275-285,1994.
Tyndall, La Mantis, Thacker, Favaloro, and Kamen, Nuc. Acids. Res., 9:6231, 1981.
Vannice and Levinson, J. Virology, 62:1305, 1988.
van Wezel, Nature, 216:64-65, 1967.
Vasseur, Kress, Montreau, and Blangy, Proc. Natl. Acad Sci. US.A., 77:1068, 1980.
Vodvotz, Bogdan, Paik, Xie, Nathan, J. Exp. Med. , 178:605, 1993.
?S Vose and White, Cancerlmmunol. Immunother., 15:227-234,1983.
Walsh, Nienhuis, Samulski, Brown, Miller, Young, and Liu, Proc. Nalt. Acad.
Sci. USA, 89:7257-7261, 1994; J. Clin. Invest., 94:1440-1448, 1994.

Wang and Calame, Cell, 47:241, 1986.
Weber, De Villiers, and Schaffner, Cell, 36:983, 1984.
Wei, Wei, Samulski, and Barranger, Gene Therapy, 1:261-268, 1994.
Weidner N, Semple JP, Welch JR, Folkman J., N Engl J Med 1991;324:1-8.
Weidner N, Carroll PR, Flax J, Flumenfeld W, Folkm an J., Am J Pathol 1993;143 :401-409.
Weinberger, Jat, and Sharp, Mol. Cell. Biol., 8:988, 1!84.
Weisz, Oguchi, Cicatiello, Esumi, J. Biol. Chem., 269:8324, 1994.
Welgus, Campbell, Bar-Shavit, Senior, Teitelbaum, J. Clin. Invest., 76:219-230, 1985.
Welgus, Campbell, Cury, Eisen, Senior, Wilhelm, Goldberg, J. Clin. Invest., 86:1494, 1990.
Welgus, Connolly, Senior, J. Clin. Invest., 77:1675, 1986.
Welgus, Senior, Parks, Kahn, Ley, Shapiro, Campbell, Matrix, (Suppl. I), 363-367, 1992.
Werb and Gordon, J. Exp. Med., 142:361-377, 1975.
Werb, Textbook of Rheumatolo~, 3rd Ed., W. N. :Kelly, E. D. Harris, S. Ruddy, and C. B.
Sledge, eds., W.B. Saunders, Philadelphia, 300, 1989.
Werb, Banda, Takemura, Gordon, Handbook of Experimental Immunology, Vol. 2., Cellular Immunology, D. M. Weir, C. Blackwell, ~ and L. A. Herzenberg, eds., Blackwell Scientific, Oxford, pp. 47.21-47.24, 1986.
White and Kuhn, J. Exp. Med., 146:802, 1977.
Whitworth, Pak, Esgro, Kleinerman, Fidler, Cancer lLletastasis Rev., 8:319-351, 1990.
WilIiams, McCIanahan, and Morimoto, Mol. Cell. Biol., 9:2574, 1989.
Winoto and Baltimore, Cell, 59:649, 1989.
Woessner, Jr., FASEB J., 5:2146, 1991.
Xie, Dong, Fidler, J. Immunol., 152:3637-3644, 1994.
Yang, Chen, Trempe, J. Virol., 68:4847-4856, 1994.
2~ Yei, Mittereder, Wert, Shitsett, Wilmott, Trapnell, Hum. Gene Ther., 5:731-744,1994.
Yoder, Kang, Zhou, Luo, and Srivastava, Blood, 82 (Supp.): 1:347A, 1994.
Young et al., N. Engl. J. Med., 299:1261-1266, 1978.
Young, Aquino, Young, J. Leukoc. Biol., 45:262, 1980.

Young, Lozano, Googan, Wright, Young, Bagash, Int. J. Cancer, 50:628-634, 1992.
Yuan et al., P.N.A.S., 89:8006-8010, 1992.
Yuan and Altman, Science, 263:1269-1273, 1994.
Yutzey, Kline, and Konieczny, Mol. Cell. Biol., 9:1397, 1989.
Zhang, Alley, Russell, Morrison, Infect. Immunol., 62:33-40,1994a.
Zhang, Mazur, French, Georges, Roth, Cancer Gene Ther., 1:5-13,1994b.
Zhou, Broxmyer, Cooper, Harrington, and Srivastava, Exp. Hematol. (NY), 21:928-933, 1993.
Zhou, Cooper, Kang, Ruggieri, Heimfeld, Srivastava, and Broxmeyer, J. Exp.
Med. , 179:1867-1875, 1994.

SEQUENCE LISTING
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Claims (68)

CLAIMS:

1. A composition comprising a .beta.-interferon inhibitory factor and a DNA
segment.

2. The composition of claim 1, wherein said .beta.-interferon inhibitory factor is an anti-.beta.-interferon antibody.

3. The composition of claim 1, wherein said .beta.-interferon inhibitory factor is an antisense .beta.-interferon nucleic acid.

4. The composition of claim 1, wherein said .beta.-interferon inhibitory factor is a .beta.-interferon-specific ribozyme.

5. The composition of claim 1, wherein said .beta.-interferon inhibitory factor is a kinase inhibitor.

6. The composition of any preceding claim, wherein said composition comprises two distinct .beta.-interferon inhibitory factors.

7. The composition of any preceding claim, wherein said DNA segment is operatively attached to a promoter.

8. The composition of any preceding claim, wherein said DNA segment is operatively attached to a eukaryotic promoter.

9. The composition of any preceding claim, wherein said DNA segment is operatively attached to a viral promoter.

10. The composition of any preceding claim, wherein said DNA segment comprises at least a first isolated expression unit.

11. The composition of any preceding claim, wherein said DNA segment comprises at least a first isolated coding region encoding a selected protein or peptide.

12. The composition of any preceding claim, wherein said DNA segment comprises an isolated coding region encoding a selected therapeutic protein or peptide.

13. The composition of any preceding claim, wherein said DNA segment comprises an isolated coding region encoding a tumor cell cytotoxic protein or peptide.

14. The composition of any preceding claim, wherein said DNA segment comprises an isolated coding region encoding a GM-CSF protein or peptide.

15. The composition of any preceding claim, wherein said DNA segment comprises an isolated coding region encoding an elastase protein or peptide.

16. The composition of any one of claims 1 to 10, wherein said DNA segment comprises an isolated expression unit operatively positioned in reverse orientation under the control of a promoter that directs the expression of an antisense transcript.

17. The composition of any preceding claim, wherein said DNA segment is an expression vector.

18. The composition of any preceding claim, wherein said DNA segment is a viral expression vector.

19. The composition of any preceding claim, wherein said DNA segment is an adenoviral expression vector.

20. The composition of any preceding claim, wherein said DNA segment is a replication defective adenoviral expression vector.

21. The composition of any preceding claim, wherein said DNA segment is a retroviral expression vector.

22. The composition of any preceding claim, wherein said DNA segment is a viral expression vector comprised within a recombinant virus.

23. The composition of any preceding claim, wherein said DNA segment is an adenoviral expression vector comprised within a recombinant adenovirus.

24. The composition of any preceding claim, wherein said composition is dispersed in a pharmaceutically acceptable carrier.

25. The composition of any preceding claim, wherein said composition is comprised within a host cell.

26. The composition of claim 25, wherein said composition is comprised within a tumor cell.

27. The composition of claim 25, wherein said composition is comprised within a macrophage.

28. The composition of any one of claims 1 to 24, for use in rendering a cell susceptible to DNA uptake.

29. The composition of any one of claims 1 to 24, for use in sensitising a cell to viral DNA
uptake.

30. Use of a composition in accordance with any one of claims 1 to 24 in the preparation of a cell transduction/cell infection formulation.

31. The composition of any one of claims 1 to 24, for use in transducing a cell with said DNA segment.

32. Use of a composition in accordance with any one of claims 1 to 24 in the preparation of a transducing formulation for providing a DNA segment to a cell.

33. The composition of any one of claims 1 to 24, for use in transducing a tumor cell with a tumor cell cytotoxic DNA segment.

34. Use of the composition of any one of claims 1 to 24 in the preparation of a medicament for treating cancer.

35. A cell in which the expression of .beta.-interferon is inhibited.

36. The cell of claim 1, comprising a composition in accordance with any one of claims 1 to 24.

37. The cell of claim 1 or 36, wherein said cell is a macrophage.

38. The cell of claim 1 or 36, wherein said cell is a tumor cell.

39. The cell of any one of claims 1 to 38, wherein said cell comprises an exogenous DNA
segment that is integrated into the genome in operable relation to a promoter in the genome.

40. The cell of any one of claims 1 to 38, wherein said cell comprises an exogenous DNA
segment that is provided to said cell operatively positioned under the control of an exogenous promoter.

41. The cell of any one of claims 1 to 40, for use in the formulation of an anti-cancer therapeutic.

42. Use of the cell of any one of claims 1 to 40 in the manufacture of a medicament for treating cancer.

43. A method of providing a DNA segment to a cell, comprising contacting said cell with a composition comprising a .beta.-interferon inhibitory factor at an amount and a time effective to increase the cell's susceptibility to DNA uptake, and providing a DNA segment to said cell.

44. The method of claim 43, wherein said cell is contacted with said .beta.-interferon inhibitory factor at a time from about 24 hours before provision of said DNA segment to about 48 hours after provision of said DNA segment.

45. The method of claim 43 or 44, wherein said cell is contacted with said .beta.-interferon inhibitory factor at a time simultaneously with provision of said DNA segment to about 24 hours after provision of said DNA segment.

46. The method of any one of claims 43 to 45, wherein said cell is contacted with a composition in accordance with any one of claims 1 to 24.

47. The method of any one of claims 43 to 46, wherein said cell is a macrophage.

48. The method of any one of claims 43 to 46, wherein said cell is a tumor cell.

49. The method of any one of claims 43 to 48, wherein said cell is located within an animal.

50. The method of claim 49, wherein said animal is a human subject.

51. Use of a composition comprising a GM-CSF or elastase protein, peptide or nucleic acid in the manufacture of a medicament for treating cancer.

52. A method of inhibiting a tumor in an animal, comprising providing to said tumor a therapeutically effective amount of GM-CSF or elastase protein, peptide or nucleic acid.

53. The method of claim 52, wherein said tumor is provided with a GM-CSF or elastase protein or peptide.

54. The method of claim 52, wherein said tumor is provided with a GM-CSF or elastase nucleic acid.

55. The method of claim 52, wherein said tumor is provided with a GM-CSF
protein, peptide or nucleic acid.

56. The method of claim 52, wherein said tumor is provided with an elastase protein, peptide or nucleic acid.

57. The method of claim 52, wherein said tumor is provided with a GM-CSF
protein, peptide or nucleic acid and an elastase protein, peptide or nucleic acid.

58. The method of any one of claims 52 to 58, wherein said tumor is further provided with a .beta.-interferon inhibiting factor.

59. The method of any one of claims 52 to 58, wherein said tumor is provided with a recombinant vector that expresses at least one of a GM-CSF or elastase protein or peptide.

60. The method of any one of claims 52 to 59, wherein said tumor is provided with said recombinant vector by contacting said tumor with a host cell that comprises said vector.

61. The method of claim 60, wherein said host cell that comprises said vector is a cell in which the expression of .beta.-interferon has been inhibited.

62. The method of claim 61, wherein said host cell comprises a composition in accordance with any one of claims 1 to 24.

63. The method of any one of claims 60 to 62, wherein said host cell is removed from said animal, contacted with said vector, and returned to said animal.

64. The method of claim 60, wherein said host cell is a tumor infiltrating lymphocyte.

65. The method of claim 60, wherein said host cell is a macrophage.

66. The method of claim 60, wherein said host cell is a tumor cell.

67. The method of any one of claims 52 to 66, wherein said GM-CSF or elastase protein, peptide or nucleic acid is provided to said tumor via parenteral, intravenous, subcutaneous or oral administration.

68. The method of any one of claims 60 to 67, wherein said animal is a human subject.