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WO1998039449A9 - COMPOSITIONS ET UTILISATIONS DE M-CSF-alpha - Google Patents

COMPOSITIONS ET UTILISATIONS DE M-CSF-alpha

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Publication number
WO1998039449A9
WO1998039449A9 PCT/US1998/004802 US9804802W WO9839449A9 WO 1998039449 A9 WO1998039449 A9 WO 1998039449A9 US 9804802 W US9804802 W US 9804802W WO 9839449 A9 WO9839449 A9 WO 9839449A9
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Prior art keywords
csfα
csf
cells
vector
mutant
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PCT/US1998/004802
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English (en)
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WO1998039449A1 (fr
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Priority to EP98910322A priority Critical patent/EP0973904A1/fr
Priority to JP53892498A priority patent/JP2001517079A/ja
Priority to AU64588/98A priority patent/AU6458898A/en
Publication of WO1998039449A1 publication Critical patent/WO1998039449A1/fr
Publication of WO1998039449A9 publication Critical patent/WO1998039449A9/fr

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  • the present invention relates to immune augmentation. Specifically, the invention relates to M-CSF ⁇ mutants having a decreased capacity to be proteolytically processed and released from a cell membrane, as delivered in a gene therapy protocol, and to methods of using these mutants to reduce a population of diseased cells.
  • the method can also optionally include combination therapeutic agents and co-administration protocols.
  • a combination therapeutic agent of the invention can include a mutant or native M-CSF ⁇ in a gene delivery vehicle, for example, with one or all of the following: soluble M-CSF polypeptide, an M-CSF ⁇ convertase inhibitor, or a prodrug activator and the prodrug.
  • Cancer in general, represents a class of diseases that has been difficult to treat.
  • primary solid tumors can generally be treated by surgical resection, a substantial number of patients who have solid tumors also possess micrometastases beyond the primary tumor site. If treated with surgery alone, many of these patients will experience recurrence of the cancer. Therefore, in addition to surgery many cancers are now also treated with cytotoxic chemotherapeutic drugs (e.g., vincristine, vinblastine, cisplatin, methotrexate, 5-FU, etc.) and/or radiation therapy.
  • cytotoxic chemotherapeutic drugs e.g., vincristine, vinblastine, cisplatin, methotrexate, 5-FU, etc.
  • Radiotherapeutic and chemotherapeutic agents are toxic to normal tissues, and often create life-threatening side effects.
  • these approaches often have extremely high failure rates of up to 90%, depending upon the type of cancer.
  • Other therapies have been attempted, in an effort to bolster or augment an individual's immune system to eliminate cancer cells.
  • Several such therapies have utilized bacterial or viral components as adjuvants, in order to stimulate the immune system to destroy the tumor cells.
  • Such components include BCG, endotoxin, mixed bacterial vaccines, interferons ( ⁇ , ⁇ , and ⁇ ), interferon inducers (e.g., Brucella abortus, and various viruses), and thymic factors (e.g., thymosin fraction 5, and thymosin alpha- 1) (see generally "Principles of Cancer Biotherapy,” Oldham (ed.), Raven Press, New York, 1987).
  • interferons ⁇ , ⁇ , and ⁇
  • interferon inducers e.g., Brucella abortus, and various viruses
  • thymic factors e.g., thymosin fraction 5, and thymosin alpha- 1
  • Such agents have generally been useful as adjuvants and as nonspecific stimulants in animal tumor models, but have not yet proved to be generally effective in humans.
  • Lymphokines have also been utilized in the treatment of cancer. Briefly, lymphokines are secreted by a variety of cells, and generally have an effect on specific cells in the generation of an immune response. Examples of lymphokines include interleukins (IL)-l, -2, -3, and -4, as well as colony stimulating factors such as G-CSF, GM-CSF, and M-CSF.
  • IL-2 interleukins
  • G-CSF GM-CSF
  • M-CSF colony stimulating factors
  • One group has utilized IL-2 to stimulate peripheral blood cells in order to expand and produce large quantities of cells that are cytotoxic to tumor cells (Rosenberg et al, N. Engl. J. Med. 575:1485-1492, 1985).
  • Phase II human clinical trials of recombinant M-CSF-beta plus anti tumor monoclonal antibody showed no antitumor responses against metastatic gastrointestinal cancer as described in Saleh et al, Cancer Res. 55:4339 (1995).
  • Macrophage colony-stimulating factor (M-CSF or CSF-1) is a cytokine that can be produced in many different forms in human cells as a result of differential mRNA splicing and variable post-translational processing, as described and illustrated in Stanley, THE CYTOKINE HANDBOOK, Chapter 21 (see fig. 1), Academic Press (1992).
  • the short clone M-CSF ⁇ is very slowly released remaining as a cell surface membrane- associated form with a half-life of about 11 hours.
  • M-CSF ⁇ is not obligately membrane-bound, and large amounts of active released M-CSF ⁇ can be recovered from virally transfected mammalian cells as described in Halenbeck et al., J. Biotechnol. 5:45 (1988). Stein et al, Blood 7 :1308 (1990) also showed that M-CSF ⁇ is active in a membrane associated form.
  • M-CSF multi-drug resistant ovarian cancer cell animal model when used in conjunction with anti-MDR antibody, as described in Sone et al, Jpn. J.
  • An embodiment of the invention is a method of reducing a population of diseased cells, the method comprising administration of a gene delivery vehicle capable of expressing an M-CSF ⁇ mutant.
  • Another embodiment of the invention is a therapeutic composition for reducing a population of diseased cells comprising a gene delivery vehicle capable of expressing an M-CSF ⁇ mutant, and a pharmaceutically acceptable carrier.
  • Another embodiment of the invention is a method of reducing a population of diseased cells comprising administration of a gene delivery vehicle capable of expressing an M-CSF ⁇ polypeptide and a pro-drug activator polypeptide, and further comprising administration of a pro-drug capable of activation by the pro-drug activator.
  • Another embodiment of the invention is a therapeutic composition for reducing a population of diseased cells comprising a gene delivery vehicle capable of expressing an M-CSF ⁇ polypeptide and a pro-drug activator polypeptide, and a pro-drug capable of activation by the pro-drug activator, and a pharmaceutically acceptable carrier.
  • Novel compositions and methods for treatment of diseases, which manifest populations of diseased cells in the patient and for which the patient has an insufficient immune response are described.
  • the diseases treatable include cancer and Leishmania, for example, among others.
  • the method involves introduction and expression of a polynucleotide encoding a mutant M-CSF ⁇ in cells from a population of diseased cells.
  • the M-CSF ⁇ is mutated so that the expressed polypeptide remains in the cell membrane longer than a non-mutated (or wild type) form of M-CSF ⁇ .
  • Expression of the mutated M-CSF ⁇ in the population of cells may provide an animal with an increased immune response against the diseased cells.
  • expression of mutant M-CSF ⁇ in tumor cells provides an increase in an anti-tumor response in the patient and may provide long-term immunity.
  • the invention may be useful for treatment in situ by direct injection or treatment by reintroduction of DNA-transformed diseased cells to the patient as a vaccine following DNA transformation.
  • Efficacy of the treatment may be improved by co-administration or subsequent administration of the M-CSF ⁇ mutant polynucleotide with cytokine administration, for example, administration of a soluble M-CSF, and also co-administration of a compound capable of inhibiting the release of M-CSF ⁇ from the cell membrane.
  • an inhibitor capable of inhibiting the release of M-CSF ⁇ from the membrane is administered, optionally, wild type M-CSF ⁇ can be administered to the patient.
  • This aspect of the invention is based on the fact that membrane-bound M-CSF ⁇ may be released by a convertase enzyme. An inhibitor of this enzyme would facilitate retention of M-CSF ⁇ in the plasma membrane.
  • Administration of the mutant M-CSF ⁇ polynucleotide can also be facilitated by administration in a gene therapy vector also expressing thymidine kinase (TK), or by co-administration with a vector capable of expressing a pro-drug activator, for example herpes simplex virus - thymidine kinase (HSV-TK).
  • TK thymidine kinase
  • HSV-TK herpes simplex virus - thymidine kinase
  • the polynucleotides of the invention can be administered in a viral vector, including for example a retroviral vector, or in a non- viral vector, including for example administration as naked DNA with an expression control sequence for intracellular expression.
  • FIG. 1 is a schematic representation of the plasmid pKm201TK-MCSF, a plasmid capable of expressing membrane-associated M-CSF, also known as M-CSF ⁇ , and also capable of expressing thymidine kinase (TK).
  • This plasmid contains the herpes simplex virus thymidine kinase (TK) gene followed by the encephalomyocarditis virus (ECMV) internal ribosome entry site (IRES) sequence, and the human mMCSF gene (also known as M-CSF ⁇ ).
  • the IRES sequence is from the 5' untranslated region of ECMV and allows cap-independent translation as described in Morgan et al, Nucl Acids Res. 20:1293-1299. The IRES allows both genes to be expressed by a single promoter.
  • This plasmid also contains AAV ITRs for packaging into recombinant AAV vectors.
  • FIG. 2 is a schematic diagram of several hydroxamate candidate therapeutic M- CSF ⁇ convertase inhibitors.
  • FIG. 2a is a British Biotech compound described in Nature 370:555 (1994);
  • FIG. 2b is RO 319790;
  • FIG. 2c is GL 129471; and
  • FIG. 2d is TAP-1.
  • GDV gene delivery vehicle
  • the cell can be inside the patient, as in in vivo gene therapy, or can be removed from the patient for transfection and returned to the patient for expression of the polypeptide as in ex vivo gene therapy.
  • the gene delivery vehicle can be any component or vehicle capable of accomplishing the delivery of a gene to a cell, for example, a liposome, a particle, or a vector.
  • a gene delivery vehicle is a recombinant vehicle, such as a recombinant viral vector, a nucleic acid vector (such as plasmid), a naked nucleic acid molecule such as genes, a nucleic acid molecule complexed to a polycationic molecule capable of neutralizing the negative charge on the nucleic acid molecule and condensing the nucleic acid molecule into a compact molecule, a nucleic acid associated with a liposome
  • the desirable properties include the ability to express a desired substance, such as a protein, enzyme, or antibody, and/or the ability to provide a biological activity, which is where the nucleic acid molecule carried by the gene delivery vehicle is itself the active agent without requiring the expression of a desired substance.
  • Gene delivery vehicle refers to an assembly that is capable of directing the expression of the sequence(s) or gene(s) of interest.
  • the gene delivery vehicle will generally include promoter elements and may include a signal that directs polyadenylation.
  • the gene delivery vehicle includes a sequence which, when transcribed, is operably linked to the sequence(s) or gene(s) of interest and acts as a translation initiation sequence.
  • the gene delivery vehicle may also include a selectable marker such as Neo, SV2 Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, as well as one or more restriction sites and a translation termination sequence.
  • Gene delivery vehicles as used within the present invention refers to recombinant vehicles, such as viral vectors (Jolly, Cancer Gen. Therapy 7:51-64, 1994), nucleic acid vectors, naked DNA, cosmids, bacteria, and certain eukaryotic cells (including producer cells; that are capable of eliciting an immune response within an animal.
  • a "population of diseased cells” is a population of cells that may or may not have inappropriate antigen expression and that may or may not be eluding the immune system attack in the patient. Diseased cells may result from pathogenic infection of the cells, including viral infection, hyperproliferation, or other abnormality contributing to the formation of a population of cells believed to be harmful to the patient. It may be said that the diseased cells express a "foreign antigen".
  • a foreign antigen is an antigen that is not normally expressed at significant levels in cells of the post embryonic host organism. For example, in a patient with cancer, the cancer cells in the patient express foreign antigens, which are not present at significant levels in the normal post-embryonic cells of the patient. Another example of cells expressing foreign antigens is cells, which are virally infected, or infected with a parasitic organism.
  • a "cytokine” refers to a group of secreted proteins that regulate the intensity and duration of an immune response by stimulating or inhibiting the proliferation of various immune cells or their secretion of antibodies or other cytokines, as described in Kuby, IMMUNOLOGY, (W.H. Freeman & Co., NY 1992). Cytokines that can increase a CD4 + T-cell count in a patient include, for example, IL-2, IL-4, IL-7, IL-9, IL-12, IL-15, and gamma interferon ( ⁇ LNF), some of which are described in Kuby, IMMUNOLOGY (W.H., Freeman & Co., NY 1992) pp. 249 and 252-253.
  • IL-1 IL-2
  • IL-3 IL-4
  • IL-4 Tepper et al,Cell 57:503-512, 1989
  • Golumbek et al Science 254:713-716, 1991
  • GM-CSF WO 85/04188
  • M-CSF Mofson et al, Cell Immunol 119: 182 (1989) which describe long clone secreted M-CSF, and Cerretti et al. Mol. Immunol. 25:761 (1988), which describes the three basic clones of M-CSF. Wing et al, J. Immunol. 135:2052 (1985), Suzu et al, Cancer Res. 49:5013 (1989)), tumor necrosis factors (TNFs) (Jayaraman et al, J.
  • Pathogenic agent refers to a cell that is responsible for a disease state.
  • pathogenic agents include tumor cells, autoreactive immune cells, hormone secreting cells, cells which lack a function that they would normally have, cells that have an additional inappropriate gene expression which does not normally occur in that cell type, and cells infected with bacteria, viruses, or other intracellular parasites.
  • pathogenic agent may also refer to a cell which over-expresses or inappropriately expresses a retroviral vector (e.g., in the wrong cell type), or which has become tumorigenic due to inappropriate insertion into a host cell's genome.
  • the therapeutic agents of the invention include polynucleotides encoding M- CSF ⁇ mutants, polynucleotides encoding native M-CSF ⁇ , soluble M-CSF polypeptides, compounds capable of inhibiting proteolytic processing and release of M-CSF ⁇ from a cell membrane, a gene delivery vehicle capable of expressing an M-CSF ⁇ mutant, a gene delivery vehicle capable of expressing a pro-drug activator polypeptide, and the pro-drug that it activates.
  • a compound capable of inhibiting proteolytic processing and release of M-CSF ⁇ from a cell membrane can also be called an M-CSF ⁇ convertase inhibitor, where M- CSF ⁇ convertase is a molecule capable of proteolytic processing and release of M-CSF ⁇ from a cell membrane.
  • a “therapeutic agent” as used herein can be any agent that accomplishes or contributes to the accomplishment of one or more of the therapeutic goals of the invention.
  • the therapeutic agent is a polynucleotide designed to express a membrane-associated M-CSF ⁇
  • that agent will be a polynucleotide that can be administered to and expressed in a cell in the patient.
  • the active form of the agent will initially be the expressed M-CSF ⁇ polypeptide in the cell membrane, and in addition release of a small amount of soluble M-CSF ⁇ may act as a chemoattractant for macrophages which can target the tumor cell, although the invention is not limited to any theories of mechanism.
  • a therapeutic agent will achieve a therapeutic goal, alone or in combination with other agents, for example, the use of an M-CSF ⁇ expressed in a cell in combination with an inhibitor of an M-CSF ⁇ convertase, can provide longer presence of the M-CSF ⁇ in the cell membrane or a transient accumulation, and thus increase the therapeutic effects of the therapy.
  • the therapeutic agents that act as M- CSF ⁇ convertase inhibitors can be, for example, a small organic molecule, a peptide, a peptoid (defined below), a polynucleotide, a polypeptide, or a nucleoside.
  • the therapeutic agent that can act as a chemoattractant for a cell expressing an M-CSF ⁇ can include, for example, a soluble M-CSF administered in polypeptide form, or an M-CSF ⁇ that is admimstered as a polynucleotide for expression in and secretion from a cell in the patient.
  • Macrophage-colony stimulating factor ⁇ or M-CSF ⁇ is a 256 amino acid protein as defined by amino acid -32 and ending at amino acid 224, where #1 amino acid is the N-terminus of the processed mature native protein.
  • M-CSF ⁇ includes all muteins, variants, analogs, and derivatives of M-CSF ⁇ that have bioactivity, whether or not the polypeptide derivative is anchored in the membrane of a cell.
  • a wild type M- CSF ⁇ is described in Kawasaki U.S. Patent No. 4,847,201, Rettenmeir et al, Mol. Cell. Biol 7:2378-2387 (1987), Halenbeck et al, J.
  • M-CSF ⁇ is a homo or heterodimer of M-CSF that is linked by a disulfide bond.
  • An example of a DNA encoding the wild-type sequence of M-CSF ⁇ is SEQ ID No. 1, and the polypeptide it encodes is exemplified in SEQ ID No. 2.
  • a "M-CSF ⁇ mutant" is any mutated form of M-CSF ⁇ .
  • An M-CSF ⁇ mutant can be generated by deletion, substitution, or addition of an amino acid as compared to a wild type M-CSF ⁇ .
  • Mutant M-CSF ⁇ can be made starting from wild type M-CSF ⁇ , for example, M-CSF ⁇ wild type as described in Kawasaki et al, Science 230:291-296 (1988), or wild type M-CSF ⁇ in plasmid pKm201TK-mMCSF deposited with the ATCC, having ATCC No. 98335.
  • An M-CSF ⁇ mutant having a decreased capacity to be proteolytically processed and released from the cell membrane is an altered form of M-CSF ⁇ such that the altered M-CSF ⁇ polypeptide has a decreased capacity to be proteolytically processed and released from the cell membrane.
  • the rate of proteolytic processing and release can be determined experimentally using techniques known to those of skill in the art. For example, cells can be transfected with a polynucleotide sequence encoding an altered or mutant M-CSF ⁇ . Supernatants and pellets from the transfected cells can be collected and subjected to quantitation of M-CSF.
  • Any mutant that demonstrates a 30% reduction and preferably at least a 50% reduction in release of the membrane- bound M-CSF into the media when compared to the wild type M-CSF ⁇ determined as described in Deng et al., J. Biol. Chem. 277:16338-16343 (1996) will be considered to be an M-CSF ⁇ mutant having decreased capacity to be proteolytically processed and released from the cell membrane.
  • a rabbit polyclonal antiserum raised against recombinant M-CSF can be used in the Western blot analysis such as described in Halenbeck et al, J. Biotechnology 5:45-58 (1988) to estimate M-CSF levels.
  • the amount of M-CSF released into the medium can also be measured with a commercially available ELISA (available from R&D, Inc. located in Minneapolis, MN) following manufacturer's protocol.
  • the invention may work as follows.
  • the membrane-bound M-CSF ⁇ may function to bind macrophages that are drawn by a gradient of the small amount of released M-CSF (a known macrophage attractant) and be bound to the tumor via the M-CSF receptor, c- fms, becoming activated in the process.
  • the activated macrophages may then generate a bystander effect as described in Ramesh et al,Exp. Hematol.
  • the instant invention controls the ratio of membrane-bound M-CSF ⁇ to released M-CSF. This is done, for example, by mutating or deleting the amino acids at or near the known M-CSF ⁇ cut site domain (described in Halenbeck et al, J. Biotechnol 5:45 (1988)), to increase the amount of membrane-bound M-CSF ⁇ .
  • the inventors also contemplate including a smaller amount of M-CSF ⁇ in the DNA transfection of the mutant M-CSF ⁇ or supplying soluble M-CSF (of any form including for example, mature M-CSF ⁇ , M-CSF ⁇ , and M-CSF ⁇ ), as described in U.S. Patent No. 5,422,105 which discloses use of M-CSF to treat tumor burden.
  • native M-CSF ⁇ can be used in conjunction with an inhibitor of the M-CSF ⁇ convertase responsible for the slow release of M-CSF ⁇ , also called M-CSF ⁇ convertase. Co-administration of an inhibitor of the convertase with the transfected cells may cause the M-CSF ⁇ to remain in the cell membrane longer than it would without the inhibitor.
  • an M-CSF ⁇ mutant can be a protein that is altered by a deletion, substitution or addition from wild type M-CSF ⁇ and as a result of the alteration the M- CSF ⁇ mutant resulting can have a decreased capacity to be proteolytically processed and released from a cell membrane or a plasma membrane.
  • an M-CSF ⁇ mutant can be the polypeptide encoded by the polynucleotide sequence of SEQ ID No. 3, and the polypeptide mutant can have the sequence of SEQ ID No. 4.
  • Proteolytic processing and release from a cell membrane in the case of a protein like M-CSF ⁇ that after translation is directed to the membrane of a cell refers to the complete solubilization of the transmembrane protein so that it is released from the cell and enters the fluid and substance outside the cell, thereby leaving the cell.
  • M-CSF ⁇ is placed in the membrane and remains there for a variable period of time.
  • M-CSF ⁇ is then released from the cell membrane by a proteolytic cleavage event that is accomplished by an M-CSF ⁇ convertase, and as a result of this proteolytic cleavage by the convertase, the M-CSF ⁇ is released into the extracellular region of the cell.
  • An M- CSF ⁇ mutant having a decreased capacity to be proteolytically processed and released from a cell membrane or plasma membrane as compared to the wild type M-CSF ⁇ means that the M-CSF ⁇ mutant remains on the membrane longer than the wild type would under the same conditions.
  • An M-CSF ⁇ mutant having a decreased capacity to be proteolytically processed and released from a cell membrane may retain other bioactivity of the wild type M-CSF ⁇ , even while having a decreased capacity to be proteolytically processed and released from a cell membrane or plasma membrane.
  • M-CSF ⁇ mutants of the invention have a bioactivity characterized by an ability to remain in the cell membrane longer than wild type M-CSF ⁇ .
  • an M-CSF ⁇ mutant of the invention can have, for example, a deletion, substitution or addition in the region of amino acids 147 to 165 of the wild type M-CSF ⁇ . Due to alterations in the sequence of wild type M-CSF ⁇ to form mutant M- CSF ⁇ polypeptides, other bioactivities of the polypeptide such as, for example, an ability to bind a receptor, or act as a chemoattractant, may or may not be affected.
  • the invention contemplates M-CSF ⁇ mutants that remain on the cell membrane longer than wild type M-CSF ⁇ and preferably mutants that retain an ability to bind an M-CSF ⁇ receptor or to act as a macrophage chemoattractant.
  • M-CSF ⁇ mutants that are capable of remaining in a cell membrane longer than wild type M-CSF ⁇ (also called M-CSF ⁇ mutants having a decreased capacity to be proteolytically processed and released from a cell membrane or plasma membrane) can be used to achieve a more efficacious effect from the gene therapy.
  • M-CSF ⁇ mutants having at least one amino acid deleted or substituted between amino acids 150 and 165 of a wild type M-CSF ⁇ can be used.
  • a deletion mutant of M-CSF ⁇ that has a deletion in amino acids 150 to 156, a deletion of amino acids 156 to 160, a deletion of amino acids 159-165, a deletion of amino acids 161-165, a deletion of amino acids 161 and 162, deletion of amino acids 163, 164, and 165, or a deletion of amino acid 161 can be used in a gene therapy protocol. Further a substitution of amino acids 158- 160, a substitution in the region between amino acids 150 to 156, a substitution in the region between amino acids 160 to 165, a substitution of Leul63 for He, or a substitution of Gin 164 for Pro, may also be selected for the therapy. More specifically, the substitution can be Asp for amino acids 158-160.
  • the mutant forms of M-CSF ⁇ can include, for example, those cleavage resistant forms having a decreased capacity to be proteolytically processed and released from a cell membrane that are described in Deng et al, J. Biol Chem. 277:16338 (1996), in which mutations and deletions near the M-CSF ⁇ cleavage site were evaluated for their effect on M-CSF ⁇ release from the cell membrane.
  • M-CSF ⁇ constructs containing a substitution of Asp for residues 158-160 or deletion of residues 161-165, or similar constructs not extending before residue 147 and/or significantly after residue 166 can result in reduced or essentially no release of M-CSF ⁇ , while retaining both c-fms binding activity and biological activity of both the membrane-bound M-CSF ⁇ and the small amount of M-CSF that does get cleaved.
  • an optimal ratio of membrane bound to released M-CSF may be obtained for a given tumor type.
  • a polynucleotide coding sequence refers to either RNA or DNA that encodes a specific amino acid sequence or its complementary strand.
  • a polynucleotide also may include, for example, an antisense oligonucleotide, or a ribozyme, and may also include such items as a 3' or 5' untranslated region of a gene, or an intron of a gene, or other region of a gene that does not make up the coding region of the gene.
  • the DNA or RNA may be single stranded or double stranded. Homologous sequences may be considered equivalents, and the homology can be determined by hybridization using standard techniques under stringent conditions, and may be about 70% to 85% or more homologous.
  • Synthetic nucleic acids or synthetic polynucleotides can be chemically synthesized nucleic acid sequences and may also be modified with chemical moieties to render the molecule resistant to degradation.
  • Synthetic nucleic acids can be ribozymes or antisense molecules, for example, which may be employed as inhibitors or expression or activity of a particular gene. Modifications to synthetic nucleic acid molecules include nucleic acid monomers or derivative or modifications thereof, including chemical moieties.
  • a polynucleotide can be a synthetic or recombinant polynucleotide, and can be generated, for example, by polymerase chain reaction (PCR) amplification, or recombinant expression of complementary DNA or RNA, or by chemical synthesis.
  • PCR polymerase chain reaction
  • M-CSF ⁇ mutants of the invention containing a deletion or substitution of at least one amino acid can be constructed as is standard in the art using site directed mutagenesis and cassette mutagenesis, including the use of PCR.
  • M-CSF mutant construct examples include Deng et al, J. Biol. Chem. 277:16338-16343 (1996).
  • Related methods for obtaining M-CSF ⁇ muteins are described in Kawasaki U.S. Patent No. 4,847,201 and Taylor et al, J. Biol Chem. 259:31171-31177 (1994).
  • the starting material can be some form of wild type M-CSF ⁇ , for example the wild type form of M- CSF ⁇ as described in Kawasaki et al, Science 250:291-296 (1988), or the wild type M- CSF ⁇ present in the plasmid pAAV-TK-mMCSF, ATCC No. 98335, or the wild type M- CSF ⁇ of SEQ ID No. 1.
  • deletions and substitutions including for example the construction of a polynucleotide encoding fusion proteins, can be engineered by standard molecular biology techniques. For example, kits for gene modification such as a kit called Unique Site Elimination Mutagenesis, available from Pharmacy Biotech, located in Rahway, N.J., can be used.
  • polynucleotides can be constructed and cloned as described in other conventional techniques of molecular biology, microbiology, and recombinant DNA technology that are within the skill of the art. Such techniques are explained fully in the literature. See e.g., Sambrook et al, (1989), MOLECULAR CLONING: A
  • Sequences that encode the described genes may be constructed as described above for any polynucleotide and also be synthesized, for example, on an Applied Biosystems Inc. DNA synthesizer (e.g., ABI DNA synthesizer model 392 (Foster City, California)). All publications, patents, and patent applications cited herein are incorporated by reference.
  • Another embodiment includes mutating the M-CSF ⁇ gene, for example by any of the standard methods described above, to generate forms of M-CSF ⁇ that bind the M- CSF receptor, and using these forms for gene therapy as described above. These forms may also be useful for the proposed therapy possibly also paired with subsequent activation of the patient's macrophages.
  • M-CSF ⁇ constructs with insertions following residue 146 and before approximately residue 160, said inserts designed to increase the distance of the receptor-binding region from the membrane and enhance macrophage binding.
  • some of the sequence of M-CSF ⁇ may be used.
  • SCF stem cell factor
  • flt3 ligand flt3-L
  • FM3-L and possible modifications of flt3-L are described in Lyman et al, Blood 55:2795-2801 (1994), and Lyman, et al. Oncogene 70:149-157 (1994).
  • a mutant M-CSF ⁇ , and/or a mutant SCF, both altered to stay in the cell membrane longer, might be administered at critical times during pregnancies in humans with histories of recurrent abortions.
  • polypeptides can be polypeptides.
  • a "polypeptide" of the invention includes any part of the protein including the mature protein, and further includes truncations, variants, alleles, analogs and derivatives thereof.
  • M-CSF is a dimer, and can be considered a dimer of polypeptides in its active form.
  • a polypeptide as used herein can include monomer, dimer and multiple components of polypeptides that together form an active polypeptide or protein.
  • Variants can be spliced variants expressed from the same gene as the related protein.
  • polypeptide or polypeptide dimer possesses one or more of the bioactivities of the protein, including the activities of a dimer of polypeptides.
  • This term is not limited to a specific length of the product of the gene.
  • polypeptides that are identical or contain at least 60%, preferably 70%, more preferably 80%, and most preferably 90% homology to the target protein or the mature protein, wherever derived, from human or nonhuman sources are included within this definition of a polypeptide.
  • alleles, muteins, and variants of the product of the gene that contain amino acid substitutions, deletions, or insertions.
  • amino acid substitutions can be conservative amino acid substitutions or substitutions to eliminate non-essential amino acid residues such as to alter a glycosylation site, a phosphorylation site, an acetylation site, or to alter the folding pattern by altering the position of the cysteine residue that is not necessary for function, etc.
  • Conservative amino acid substitutions are those that preserve the general charge, hydrophobicity/hydrophilicity and/or steric bulk of the amino acid substituted, for example, substitutions between the members of the following groups are conservative substitutions: Gly/Ala, Val/Ile/Leu, Asp/Glu, Lys/Arg, Asn/Gln, Ser/Cys/Thr/Ala and Phe/Trp/Tyr.
  • Analogs include peptides having one or more peptide mimics, also known as peptoids, that possess the target protein-like activity. Included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and nonnaturally occurring.
  • polypeptide also does not exclude post-expression modifications of the polypeptide, for example, glycosylations, acetylations, phosphorylations, myristoylations and the like.
  • polypeptide also includes any M-CSF ⁇ fusion protein engineered to some advantage for the therapy, for example, including a polypeptide region that improves expression, or a region that increases the M-CSF ⁇ resistance to cleavage by M-CSF ⁇ convertase.
  • any of the peptides or polypeptides of the invention can be PEGylated.
  • PEGylated refers to covalently added polymers of polyethylene glycol at one or more sites of a polypeptide that do not greatly inhibit the biological activity of the polypeptide in vitro.
  • Biologically active pegylated M-CSF can be prepared as described in U.S. 4,847,325 which is incorporated by reference in full.
  • fusion protein or “fusion polypeptide” refers to the recombinant expression of more than one heterologous coding sequence in a vector or contiguous connection such that expression of the polypeptide in the vector results in expression of one polypeptide that includes more than one protein or portion of more than one protein.
  • Fusion proteins can be called chimeric proteins. Most optimally, the fusion protein retains some or all of the biological activity of the polypeptide units from which it is built, and preferably, the fusion protein generates a synergistic improved biological activity by combining the portion of the separate proteins to form a single polypeptide.
  • a fusion protein can also be created with a polypeptide that has function and a peptide or polypeptide that has no function when expressed, but which serves a purpose for the expression of the polypeptide with activity.
  • fusion proteins useful for the invention include any M-CSF ⁇ fusion protein engineered to some advantage for the therapy, for example, including a polypeptide region that improves expression, or substition of a region to provide an increase of the M-CSF ⁇ resistance to cleavage by M- CSF ⁇ convertase.
  • a fusion protein can be made of the M-CSF ⁇ extracellular domain from amino acids 1 to 146, and the remainder of the molecule, including the proximal extracellular portion and optionally the intracellular portion of M- CSF ⁇ can be replaced with corresponding sequence from another protein from the same or a similar family, or may be replaced by the corresponding regions of another transmembrane protein that is not cleaved from the cell surface.
  • the region of M-CSF ⁇ from amino acids 147 to 170 can be replaced by the corresponding region of a transmembrane protein that is not normally cleaved from the cell surface, while retaining the transmembrane, cytoplasmic and remaining extracellular portion of the wild type M-CSF ⁇ .
  • the extracellular sequence of M-CSF ⁇ from amino acid no. 1 to about amino acid no.146, and which binds the M-CSF ⁇ receptor can be retained in the fusion protein.
  • the proximal extracellular domain (of the fusion polypeptide) corresponding to about 14 amino acids (in the native M-CSF ⁇ protein) can be a multiple proline sequence, or a sequence having mostly proline amino acids or amino acids forming a sequence that is not normally cleaved from the cell membrane by the convertase.
  • the sequence of the transmembrane portion of the fusion polypeptide can be of hydrophobic amino acids having nonpolar side chains, including for example the amino acids alanine, valine, leucine, isoleucine, proline, phenyalanine, tryptophan, and methionine.
  • the sequence of the fusion polypeptide of amino acids proximally intracellular can be amino acids with charged side chains including, for example, aspartic acid and glutamic acid which are negatively charged at pH 6.0, and lysine, arginine, and histidine which are positively charged at pH 6.0.
  • the remainder of the fusion polypeptide can be a sequence appropriate for an intracellular portion of a transmembrane polypeptide, and might preferably be a sequence of M-CSF ⁇ or of a member the same family so that any effects that the intracellular portion of the molecule may have on the biological activity desired from the M-CSF ⁇ derived polypeptide fusion might be retained.
  • An additional, and optional, therapeutic agent of the invention is soluble M-CSF, sometimes called mature M-CSF ⁇ .
  • This soluble form can be a polynucleotide encoding the polypeptide, or the polypeptide itself.
  • the M-CSF ⁇ may have deletions or substitutions from the native molecule in a manner consistent with the goals of the therapy.
  • any variation in the molecule that either enhances this bioactivity, or at least, does not interfere with that bioactivity can be considered an acceptable M-CSF ⁇ form for the purposes of the invention.
  • the M-CSF ⁇ is used in the invention in a co-administration context, or as a component of a combination therapeutic agent.
  • An M-CSF ⁇ polypeptide can be designed and engineered as described above for M-CSF ⁇ polynucleotides, or can be expressed from a cell and purified for administration. Some exemplary expression systems for these and other polypeptides of the invention follow.
  • polypeptides of the invention can be expressed in any expression system, including, for example, bacterial, yeast, insect, amphibian and mammalian systems. Expression systems in bacteria include those described in Chang et al, Nature 275:615 (1978), Goeddel et al, Nature 257:544 (1979), Goeddel et al, Nucleic Acids Res. 5:4057 (1980), EP 36,776, U.S. Patent No. 4,551,433, deBoer et al, Proc. Natl. Acad. Sci.
  • inhibitors of M-CSF ⁇ convertase activity can be used in the invention in order to prevent release of the M-CSF ⁇ from the cell membrane.
  • Such inhibitors also called compounds capable of inhibiting proteolytic processing and release of M-CSF ⁇ from a cell membrane
  • can be polynucleotides e.g., ribozymes or antisense molecules
  • small molecules such as organic small molecules, peptides or peptoids capable of entering a cell.
  • mutant constructs or the native (wild type) M-CSF ⁇ s, or functionally equivalent constructs can be rendered temporarily or essentially permanently enhanced in their ratio of membrane-bound to released M-CSF by treatment of the transfected cells with an inhibitor of M-CSF ⁇ convertase.
  • This enzyme has not been identified, but some properties of its function have been characterized.
  • the stimulation of M-CSF ⁇ release by phorbol ester is described in Stein and Rettenmeir, Oncogene 5:601 (1991) and is reminiscent of the stimulated release of other membrane-bound cytokines by matrix metalloprotease-like enzymes as described in McGeehan et al, Nature 370:558 (1994), and Mullberg et al, J. Immunol.
  • M-CSF ⁇ a form of M-CSF shown by Mullberg not to be inhibited by hydroxamates, has a different cleavage site than M-CSF ⁇ and is believed to be solubilized intracellularly by a different convertase.
  • M-CSF ⁇ convertase refers to a protease, capable of cleaving M-CSF ⁇ or related M-CSF ⁇ mutants from the membrane of a cell to produce the soluble form of M-CSF ⁇ .
  • small molecule refers to an organic molecule derived, for example, from a small molecule combinatorial chemistry library.
  • peptide and the term “peptoid” as used herein refers to a peptide or peptoid (a peptide derivative) derived, for example, from a peptide library.
  • binding pair refers to a pair of molecules capable of a binding interaction between the two molecules. Such binding interactions can mediate a cell signaling, cell-cell interactions, and intracellular biochemical processes.
  • binding pair can refer to a protein protein, protein-DNA, protein-RNA, DNA-DNA, DNA-RNA, and RNA-RNA binding interactions, and can also include a binding interaction between a small molecule, a peptoid, or a peptide and a protein, DNA, or RNA molecule, in which the components of the pair bind specifically to each other with a higher affinity than to a random molecule.
  • An example of a binding pair is the formation of a binding pair between an M-CSF ⁇ convertase and a small molecule M- CSF ⁇ convertase inhibitor, or a M-CSF ⁇ convertase substrate and a M-CSF ⁇ convertase inhibitor.
  • Specific binding indicates a binding interaction having a low dissociation constant, which distinguishes specific binding from non-specific, background, binding.
  • Inhibition of a biological interaction can be accomplished by inhibiting an in vivo binding interaction such as, for example, a DNA-protein interaction. Such inhibition can be accomplished, for example, by an inhibitor that binds the protein, or by an inhibitor that binds the DNA, in either case, thus preventing the original endogenous binding interaction, and so the biological activity that follows from it.
  • an "inhibitor of an M-CSF ⁇ convertase” refers to a compound capable of inhibiting the cleavage of membrane-bound M-CSF ⁇ .
  • it may include an antagonist of an M-CSF ⁇ convertase.
  • the M-CSF ⁇ convertase inhibitor, or the inhibitor of a cleavage event of M-CSF ⁇ can be a polynucleotide antagonist, a polypeptide antagonist (including an antibody, and also for example an intra-antibody), a peptide antagonist, or a small molecule antagonist, or derivatives or variations of these.
  • the use and appropriateness of such inhibitors for the purposes of the invention are not limited to any theories of action of the inhibitor.
  • the inhibitor can be tested for its ability to reduce the biological activity of an M-CSF ⁇ convertase in an in vivo or in vitro assay.
  • any inhibitor that provides at least 30% and more preferably 50% inhibition of release of M- CSF ⁇ from the cell membrane as compared to non-inhibited control cells expressing M- CSF ⁇ indicates a functional inhibitor.
  • the inhibitor in the context of treatment of persons with cancer or other diseases that can be effectively treated by administration of M-CSF ⁇ , can be a hydroxamic acid inhibitor, or hydroxamate.
  • Hydroxamate inhibitors have been developed by pharmaceutical companies to inhibit other convertases, for example Glaxo (inhibitor designation GL 129471), FIG. 2c, as described in Gearing et al, Nature 370:555 (1994); Roche Products Ltd. (inhibitor designation RO 31-4742), as described in Nixon et al, Int. J. Tissue React. 13:237-43 (1991) and Finch-Arietta et al, Agents Actions 39 Spec. No. pCl 89-91 (1993), and RO 31-9790, FIG. 2b, as described in
  • inhibitors include inhibitors that selectively inhibit M-CSF ⁇ convertase.
  • Libraries or pools of candidate modulators, including for example, inhibitors, can be screened for activity related to M-CSF ⁇ convertase, for example, inhibitory activity.
  • the candidate modulators including inhibitors and libraries of candidate inhibitors can be derived from any of the various possible sources of candidates, such as for example, libraries of peptides, peptoids, small molecules, polypeptides, antibodies, polynucleotides, antisense molecules, ribozymes, cRNA, cDNA, polypeptides presented by phage display, and in general any a molecule that may be capable of inhibiting or antagonizing M-CSF ⁇ convertase activity.
  • Candidates may be derived from almost any source of pooled libraries, naturally occurring compounds, or mixtures of compounds. Described below are some exemplary and possible sources of candidates, including synthesized libraries of peptides, peptoids, and small molecules.
  • the exemplary expression systems can be used to generate cRNA or cDNA libraries that can also be screened for the ability to modulate M-CSF ⁇ convertase activity.
  • Other sources of candidates also exist, including, but not limited to polypeptides generated by phage display.
  • the modulator of M-CSF ⁇ convertase can be a peptide.
  • a peptide library can be screened to determine which peptides function as desired.
  • a "library" of peptides may be synthesized and used following the methods disclosed in U.S. Patent No. 5,010,175 (the '175 patent) and in WO 91/17823. Briefly, one prepares a mixture of peptides, which is then screened to determine the peptides exhibiting the desired M-CSF ⁇ convertase binding or inhibitory activity.
  • a suitable peptide synthesis support for example, a resin
  • a suitable peptide synthesis support for example, a resin
  • the method described in WO 91/17823 is similar, but simplifies the process of determining which peptides are responsible for any observed M-CSF ⁇ convertase antagonism or other activity.
  • the methods described in WO 91/17823 and U.S. Patent No. 5,194,392 enable the preparation of such pools and subpools by automated techniques in parallel, such that all synthesis and resynthesis may be performed in a matter of days.
  • Peptoids polymers comprised of monomer units of at least some substituted amino acids, can act as small molecule inhibitors herein and can be synthesized as described in PCT 91/19735, so as to provide libraries of peptoids that can be screened for the desired biological activity.
  • Peptoids are easily synthesized by standard chemical methods. The preferred method of synthesis is the "submonomer” technique described by Zuckermann et al, J. Am. Chem. Soc. 114:10646-7 (1992). Synthesis by solid phase of other heterocyclic organic compounds in combinatorial libraries is also described in an application entitled "Combinatorial Libraries of Substrate-Bound Cyclic Organic Compounds" filed on June 7, 1995, herein incorporated by reference in full.
  • the selected inhibitor of M-CSF ⁇ convertase is a ribozyme
  • the ribozyme can be chemically synthesized or prepared in a vector for a gene therapy protocol including preparation of DNA encoding the ribozyme sequence.
  • the synthetic ribozymes or a vector for gene therapy delivery can be encased in liposomes for delivery, or the synthetic ribozyme can be administered with a pharmaceutically acceptable carrier.
  • a ribozyme is a polynucleotide that has the ability to catalyze the cleavage of a polynucleotide substrate.
  • Ribozymes for inactivating a portion of HIV can be prepared and used as described in Long et al, FASEB J. 7:25 (1993), and Symons, w . 7?ev. Biochem. 61:641 (1992), Perrotta et al, Biochem. 57:16, 17 (1992); and U.S. Patent No. 5,225,337, U.S.Patent No. 5,168,053, U.S. Patent No. 5,168,053 and U.S. Patent No. 5,116,742, Ojwang et al, Proc. Natl. Acad. Sci. USA 59:10802-10806 (1992), U.S. Patent No. 5,254,678 and in U.S. Patent No.
  • the hybridizing region of the ribozyme or of an antisense polynucleotide may be modified by linking the displacement arm in a linear arrangement, or alternatively, may be prepared as a branched structure as described in Horn and Urdea, Nucleic Acids Res. 77:6959-67 (1989).
  • the basic structure of the ribozymes or antisense polynucleotides may also be chemically altered in ways quite familiar to those skilled in the art.
  • Ribozymes and antisense molecules can be admimstered as synthetic oligonucleotide derivatives modified by monomeric units. Ribozymes and antisense molecules can also be placed in a vector and expressed intracellularly in a gene therapy protocol.
  • any functional convertase inhibitor cells believed to express an M-CSF ⁇ convertase (based on the ability of the cell to produce soluble M-CSF ⁇ ) can be used. These cells are placed in contact with a candidate M-CSF ⁇ convertase inhibitor. Polynucleotide candidate inhibitors can transform the cells being tested. Supernatants from the contacted or transfected cells are collected and subjected to Western blot analysis, conducted by standard techniques known in the art. Any candidate M-CSF ⁇ convertase inhibitor which can demonstrated at least a 50% reduction in release of membrane bound M-CSF into the media when compared to cells not in contact or transformed with the candidate inhibitor, will be considered to be a positive inhibitor. Further testing for ultimate effectiveness can be then performed.
  • a rabbit polyclonal antiserum raised against recombinant M-CSF can be used in a Western blot analysis to detect M-CSF as described in Halenbeck et al, J. Biotechnology 5:45-58 (1988), or the quantitation of M-CSF can be performed as described in Deng et al., J. Biol. Chem.
  • the agent contains a combination of more than one agent
  • the agent is defined as a "combination therapeutic agent" which is a therapeutic composition having several components that produce their separate effects when administered.
  • the separate effects of the combination therapeutic agent combine to result in a larger therapeutic effect, for example recovery from disease and long term survival.
  • An example of separate effects resulting from administration of a combination therapeutic agent is the combination of such effects as short-term, or long-term tumor regression, or increase of an immune response targeted to tumor cells.
  • An example of a combination therapeutic agent of this invention would be (1) a polynucleotide encoding a mutant or native M-CSF ⁇ administered in a retroviral vector, or administered in a non- viral vector as naked DNA having an expression control sequence, such agents being administered alone or followed with gancyclovir administration, and an administration of (2) a compound capable of inhibiting the cleavage activity of an M-CSF ⁇ convertase, and also including, optionally administration of (3) a soluble polypeptide form of an M- CSF, or a polynucleotide encoding a M-CSF ⁇ for expression in the cells of the patient.
  • cytokines can be administered in polypeptide or polynucleotide form with administration of M-CSF ⁇ in a gene therapy protocol for effecting arrest of cancer cell growth resulting in tumor regression.
  • a combination therapeutic agent including soluble M-CSF, or a convertase inhibitor.
  • Other components of a combination therapeutic agent, for administration of an M-CSF ⁇ polynucleotide in combination with or in a combination therapeutic agent also exist.
  • the vector constructs described herein may also direct the expression of additional non- vector derived genes.
  • the non- vector derived gene encodes a protein, such as an immune accessory molecule for aiding in an immunomodulatory effect.
  • immune accessory molecules include many cytokines, for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7 (U.S. Patent No.
  • Sequences that encode a non-vector derived gene may be readily obtained from a variety of sources.
  • plasmids which contain sequences that encode immune accessory molecules may be obtained from a depository such as the American Type Culture Collection (ATCC, Rockville, Maryland), or from commercial sources such as British Bio-Technology Limited (Cowley, Oxford England).
  • Representative sources for sequences which encode the above-noted anti-tumor agents include BBG 12 (containing the GM-CSF gene coding for the mature protein of 127 amino acids), BBG 6 (which contains sequences encoding gamma interferon), ATCC No. 39656 (which contains sequences encoding TNF), ATCC No.
  • ATCC No. 20663 (which contains sequences encoding alpha interferon), ATCC Nos. 31902, 31902 and 39517 (which contains sequences encoding beta interferon), ATCC No 67024 (which contains a sequence which encodes Interleukin-1), ATCC Nos. 39405, 39452, 39516, 39626 and 39673 (which contains sequences encoding Interleukin-2), ATCC Nos. 59399, 59398, and 67326 (which contain sequences encoding Interleukin-3), ATCC No. 57592 (which contains sequences encoding Interleukin-4), ATCC Nos. 59394 and 59395 (which contain sequences encoding Interleukin-5), and ATCC No.
  • Immunomodulatory refers to use of factors which, when manufactured by one or more of the cells involved in an immune response, or, which when added exogenously to the cells, causes the immune response to be different in quality or potency from that which would have occurred in the absence of the factor.
  • the quality or potency of a response may be measured by a variety of assays known to one of skill in the art including, for example, in vitro assays which measure cellular proliferation (e.g., ⁇ H thymidine uptake), and in vitro cytotoxic assays (e.g., which measure 51Cr release) (see, Warner et al, AIDS Res. and Human Retroviruses 7:645-655, 1991). Immunomodulatory factors may be active both in vivo and ex vivo.
  • Such factors include cytokines, such as interleukins 2, 4, 6, 12 and 15 (among others), alpha interferons, beta interferons, gamma interferons, GM-CSF, G-CSF, and tumor necrosis factors (TNFs).
  • Other immunomodulatory factors include, for example, CD3, ICAM-1, ICAM-2, LFA-1, LFA-3, MHC class I molecules, MHC class II molecules, B7.1-.3, ⁇ 2-microglobulin, chaperones, or analogs thereof.
  • a therapy including administration of M-CSF ⁇ or an M- CSF ⁇ mutant, in conjunction with a prodrug activator and a prodrug, can be immunomodulatory.
  • a prodrug system applied in conjunction with administration of M-CSF ⁇ or an M-CSF ⁇ mutant can act as a safety mechanism for the gene therapy, or can act as a combination therapeutic agent.
  • the prodrug activator for example thymidine kinase (TK)
  • TK thymidine kinase
  • a prodrug for example gancyclovir
  • gancyclovir is added to kill the cells expressing an M-CSF ⁇ along with TK. This allows the clinician a measure of control over the gene therapy.
  • the viral vector can include a gene, for example, a suicide gene, for the purpose of inactivating expression of the polynucleotide at an appropriate or necessary time.
  • the viral vector capable of expressing the polynucleotide therapeutic can also contain, for example, a thymidine kinase gene from the Herpes simplex virus.
  • Gancyclovir is administered to the patient and a cell expressing the thymidine kinase phosphorylates the gancyclovirs causing the gancyclovir to become toxic and kill the cell.
  • the expression of the polynucleotide of interest is stopped.
  • the TK/gancyclovir system is useful for inactivating the transfected cells in the patient, where, for example, it appears that the M-CSF receptor is expressed on the surface of the tumor cells.
  • the TK gancyclovir system is also administered as combination therapeutic agent, in a combination therapy protocol, for achieving tumor cell, or pathogen-infected cell killing by both the M-CSF mechanisms described earlier, and the prodrug activation provided by the TK/gancyclovir system as just described.
  • known cDNA sequences that encode conditionally cytotoxic genes or other non-vector derived genes may be obtained from cells that express or contain such sequences. Briefly, within one embodiment mRNA from a cell which expresses the gene of interest is reverse transcribed with reverse transcriptase using oligo dT or random primers. The single stranded cDNA may then be amplified by PCR (see U.S. Patent Nos. 4,683,202, 4,683,195 and 4,800,159. See also PCR Technology: Principles and Applications for DNA Amplification, Erlich (ed.), Stockton Press, 1989 all of which are incorporated by reference herein in their entirety) utilizing oligonucleotide primers complementary to sequences on either side of desired sequences.
  • a double stranded DNA is denatured by heating in the presence of heat stable Taq polymerase, sequence specific DNA primers, ATP, CTP, GTP and TTP. Double-stranded DNA is produced when synthesis is complete. This cycle may be repeated many times, resulting in a factorial amplification of the desired DNA.
  • the invention includes gene delivery vehicles capable of expressing the contemplated M-CSF ⁇ mutant coding sequences.
  • the gene delivery vehicle is preferably a viral vector and, more preferably, a retroviral, adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirus vectors.
  • the viral vector can also be an astrovirus, coronavirus, orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus, poxvirus, togavirus viral vector. See generally, Jolly, Cancer Gene Therapy 7:51-64 (1994), Kimura, Human Gene Therapy 5:845-852 (1994), Connelly, Human Gene Therapy 5:185-193 (1995), and Kaplitt, Nature Genetics 5:148-153 (1994).
  • Retroviral vectors are well known in the art and we contemplate that any known retroviral gene therapy vector is employable in the invention, including B, C and D type retroviruses, xenotropic retroviruses (for example, NZB-Xl, NZB-X2 and NZB9-1 (see O'Neill, J. Vir. 55:160, 1985) polytropic retroviruses (for example, MCF and MCF-MLV (see Kelly, J Vir. 45:291, 1983), spumaviruses and lentiviruses. See RNA Tumor Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985.
  • xenotropic retroviruses for example, NZB-Xl, NZB-X2 and NZB9-1 (see O'Neill, J. Vir. 55:160, 1985
  • polytropic retroviruses for example, MCF and MCF-MLV (see Kelly, J Vir. 45:291, 1983)
  • spumaviruses and lentiviruses See
  • portions of the retroviral vector may be derived from different retroviruses.
  • retro vector LTRs may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin of second strand synthesis from an Avian Leukosis Virus.
  • retroviral vectors are used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines.
  • Retroviral vector particles are constructed for site-specific integration into host cell DNA by incorporation of a chimeric integrase enzyme into the retroviral particle. It is preferable that the recombinant retroviral vector is a replication defective recombinant retro virus.
  • Packaging cell lines suitable for use with the above-described retrovirus vectors are well known in the art, are readily prepared (see WO 92/05266), and can be used to create producer cell lines (also termed vector cell lines or " VCLs") for the production of recombinant vector particles capable of infecting human cells.
  • the packaging cell lines are made from human parent cells (e.g., HT1080 cells) or mink parent cell lines, which eliminates inactivation in human serum.
  • Preferred retroviruses for the construction of retroviral gene therapy vectors include Avian Leukosis Virus, Bovine Leukemia, Virus, Murine Leukemia Virus, Mink- Cell Focus-Inducing Virus, Murine Sarcoma Virus, Reticuloendotheliosis Virus and Rous Sarcoma Virus.
  • Particularly preferred Murine Leukemia Viruses include 4070A and 1504A (Hartley and Rowe, J. Virol. 79:19-25, 1976), Abelson (ATCC No. VR-999), Friend (ATCC No. VR-245), Graffi, Gross (ATCC No. VR-590), Kirsten, Harvey Sarcoma Virus and Rauscher (ATCC No.
  • Retroviruses may be obtained from depositories or collections such as the American Type Culture Collection (“ATCC”) in Rockville, Maryland or isolated from known sources using commonly available techniques.
  • ATCC American Type Culture Collection
  • retroviral gene therapy vectors employable in this invention include those described in GB 2200651, EP 0415731, EP 0345242, WO 89/02468; WO 89/05349, WO 89/09271, WO 90/02806, WO 90/07936, WO 90/07936, WO 94/03622, WO 93/25698, WO 93/25234, WO 93/11230, WO 93/10218, WO 91/02805, in U.S. Patent No. 5,219,740, U.S. Patent No. 4,405,712, U.S. Patent No. 4,861,719, U.S. Patent No. 4,980,289 and U.S. Patent No.
  • adenoviral gene therapy vectors employable in this invention include those described in the above-referenced documents and in WO 94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984, WO 95/00655, WO 95/27071, WO
  • the gene delivery vehicles of the invention also include adenovirus associated virus (AAV) vectors.
  • AAV adenovirus associated virus
  • Leading and preferred examples of such vectors for use in this invention are the AAV-2 basal vectors disclosed in Srivastava, WO 93/09239.
  • Most preferred AAV vectors comprise the two AAV inverted terminal repeats in which the native D-sequences are modified by substitution of nucleotides, such that at least 5 native nucleotides and up to 18 native nucleotides, preferably at least 10 native nucleotides up to 18 native nucleotides, most preferably 10 native nucleotides are retained and the remaining nucleotides of the D-sequence are deleted or replaced with non-native nucleotides.
  • the native D-sequences of the AAV inverted terminal repeats are sequences of 20 consecutive nucleotides in each AAV inverted terminal repeat (i.e., there is one sequence at each end) which are not involved in HP formation.
  • the non-native replacement nucleotide may be any nucleotide other than the nucleotide found in the native D-sequence in the same position.
  • Other employable exemplary AAV vectors are pWP-19, pWN-1, both of which are disclosed inNahreini, Gene 124:257-262 (1993). Another example of such an AAV vector is psub201. See Samulski, J. Virol. 61:3096 (1987).
  • Another exemplary AAV vector is the Double-D ITR vector.
  • Double D ITR vector is disclosed in U.S. Patent No. 5,478,745. Still other vectors are those disclosed in Carter, U.S. Patent No. 4,797,368, and Muzyczka, U.S. Patent No. 5,139,941, Chartejee, U.S. Patent No. 5,474,935, and Kotin, PCT Patent Publication WO 94/288157. Yet a further example of an AAV vector employable in this invention is SSV9AFABTKneo, which contains the AFP enhancer and albumin promoter and directs expression predominantly in the liver. Its structure and how to make it are disclosed in Su, Human Gene Therapy 7:463-470 (1996). Additional AAV gene therapy vectors are described in U.S. Patent No. 5,354,678, U.S. Patent No. 5,173,414, U.S. Patent No. 5,139,941, and U.S. Patent No. 5,252,479. All of the above references are hereby incorporated herein by reference.
  • the gene therapy vectors of the invention also include herpes vectors.
  • Leading and preferred examples are herpes simplex virus vectors containing a sequence encoding a thymidine kinase polypeptide such as those disclosed in U.S. Patent No. 5,288,641 and EP 0176170 (Roizman), which are incorporated herein by reference.
  • herpes simplex virus vectors include HFEM/ICP6-LacZ disclosed in WO 95/04139 (Wistar Institute), pHSVlac described in Geller, Science 241:1667-1669 (1988) and in WO 90/09441 and WO 92/07945, HSV Us3::pgC-lacZ described in Fink, Human Gene Therapy 5:11-19 (1992) and HSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), and those deposited with the ATCC as accession numbers ATCC VR-977 and ATCC VR-260.
  • alpha virus gene therapy vectors may be employed in this invention.
  • Preferred alpha virus vectors are Sindbis viruses vector, Togaviruses, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Middleberg virus (ATCC VR- 370), Ross River virus (ATCC VR-373; ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Patent Nos. 5,091,309 and 5,217,879, and WO 92/10578. More particularly, those alpha virus vectors described in PCT Patent Publications WO 94/21792, WO 92/10578, WO 95/07994, U.S. Patent No. 5,091,309 and U.S.
  • Patent No. 5,217,879 are employable.
  • Such alpha viruses may be obtained from depositories or collections such as the ATCC in Rockville, Maryland or isolated from known sources using commonly available techniques.
  • alphavirus vectors with reduced cytotoxicity are used.
  • the above references and patents relating to alpha virus are hereby incorporated herein by reference.
  • DNA vector systems such as eukarytic layered expression systems are also useful for expressing the M-CSF nucleic acids of the invention. See WO 95/07994 for a detailed description of eukaryotic layered expression systems.
  • the eukaryotic layered expression systems of the invention are derived from alphavirus vectors and most preferably from Sindbis viral vectors.
  • viral vectors suitable for use in the present invention include those derived from polio virus, for example ATCC VR-58 and those described in Evans, Nature 339:385 (1989) and Sanin, J. Biol. Standardization 7:115 (1973); rhinovirus, for example, ATCC VR-1110 and those described in Arnold, J Cell Biochem (1990) L401 ; pox viruses such as canary pox virus or vaccinia virus, for example ATCC VR-111 and ATCC VR-2010 and those described in Fisher-Hoch, Proc. Natl Acad. Sci. 86:317 (1989); Flexner, Ann. NY Acad. Sci. 569:86 (1989); Flexner, Vaccine 8:17 (1990); in U.S. Patent No.
  • measles virus for example ATCC VR-67 and VR-1247 and those described in EP 0440219; Aura virus, for example, ATCC VR-368; Bebaru virus, for example, ATCC VR-600 and ATCC VR- 1240; Cabassou virus, for example, ATCC VR-922; Chikungunya virus, for example, ATCC VR-64 and ATCC VR-1241; Fort Morgan Virus, for example ATCC VR-924; Getah virus, for example, ATCC VR-369 and ATCC VR-1243; Kyzylagach virus, for example, ATCC VR-927; Mayaro virus, for example, ATCC VR-66; Mucambo virus, for example ATCC VR-580 and ATCC VR-1244; Ndumu virus, for example ATCC VR- 371; Pixuna virus, for example, ATCC VR-372 and ATCC VR-1245; Tonate virus, for example, ATCC VR-925; Triniti virus, for example, ATCC VR-469; Una virus
  • compositions of this invention into cells is not limited to the above mentioned viral vectors.
  • Other known delivery methods and media may be employed such as, for example, nucleic acid expression vectors, polycationic condensed DNA linked or unlinked to killed adenovirus alone, for example Curiel, Hum. Gene. Ther. 5:147-154 (1992) ligand linked DNA, for example, see Wu, J. Biol. Chem. 254:16985-16987 (1989), eucaryotic cell delivery vehicles cells, deposition of photopolymerized hydrogel materials, hand-held gene transfer particle gun, as described in U.S. Patent No. 5,149,655, ionizing radiation as described in U.S. Patent No.
  • Particle mediated gene transfer may be employed. Briefly, the sequence can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, as described in U.S. Patent No.
  • Naked DNA may also be employed.
  • Exemplary naked DNA introduction methods are described in WO 90/11092 and U.S. Patent No. 5,580,859. Uptake efficiency may be improved using biodegradable latex beads.
  • DNA coated latex beads are efficiently transported into cells after endocytosis initiation by the beads. The method may be improved further by treatment of the beads to increase hydrophobicity and thereby facilitate disruption of the endosome and release of the DNA into the cytoplasm.
  • Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120, WO 95/13796, WO 94/23697, WO 91/144445 and EP 524,968. As described in co-owned U.S. Application No.
  • the nucleic acid sequences encoding an M-CSF polypeptide can be inserted into conventional vectors that contain conventional control sequences for high level expression, and then be incubated with synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin.
  • synthetic gene transfer molecules such as polymeric DNA-binding cations like polylysine, protamine, and albumin, linked to cell targeting ligands such as asialoorosomucoid, insulin, galactose, lactose, or transferrin.
  • Other delivery systems include the use of liposomes to encapsulate DNA comprising the gene under the control of a variety of tissue-specific or ubiquitously-active promoters.
  • non- viral delivery suitable for use includes mechanical delivery systems such as the approach described in Woffendin et ⁇ /., Proc. Natl. Acad. Sci. USA 91(24):! 1581-11585 (1994).
  • the coding sequence and the product of expression of such can be delivered through deposition of photopolymerized hydrogel materials.
  • Other conventional methods for gene delivery that can be used for delivery of the coding sequence include, for example, use of hand-held gene transfer particle gun, as described in U.S. Patent No. 5,149,655; use of ionizing radiation for activating transferred gene, as described in U.S. Patent No. 5,206,152 and WO 92/11033.
  • Exemplary liposome and polycationic gene delivery vehicles are those described in U.S.
  • the therapeutic agents of the invention appropriate for gene therapy include the previously described polynucleotides delivered in gene therapy vehicles, for example, in those gene therapy vehicles also described below. These vehicles include capabilities to express the polynucleotides, so that, for example, naked DNA delivered in a nonviral vector contains all the components necessary for expression.
  • naked DNA refers to polynucleotide DNA for administration to a patient for expression in the patient.
  • the polynucleotide can be, for example, a coding sequence, and the polynucleotide DNA can be directly or indirectly connected to an expression control sequence that can facilitate the expression of the coding sequence once the DNA is inside a cell.
  • the direct or indirect connection is equivalent from the perspective of facilitating the expression of the DNA in the patient's cells, and merely allows the possibility of the inclusion of other sequences between the regulatory region and the coding sequence that may facilitate the expression further, or may merely act a linker or spacer to facilitate connecting the two polynucleotide regions together to form a nonviral vector.
  • the polynucleotides of the invention can be assembled into vector constructs useful in a therapeutic context.
  • a "vector construct” refers to an assembly that is capable of directing the expression of the sequence(s) or gene(s) of interest.
  • the vector construct must include transcriptional promoter/enhancer or locus defining element(s), or other elements which control gene expression by other means such as alternate splicing, nuclear RNA export, post-translational modification of messenger, or post- transcriptional modification of protein.
  • the vector construct must include a sequence which, when transcribed, is operably linked to the sequence(s) or gene(s) of interest and acts as a translation initiation sequence.
  • the vector construct includes a signal that directs polyadenylation, a selectable marker such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR, as well as one or more restriction sites and a translation termination sequence.
  • a selectable marker such as Neo, TK, hygromycin, phleomycin, histidinol, or DHFR
  • the vector construct must include a packaging signal, long terminal repeats (LTRs), and positive and negative strand primer binding sites.
  • LTRs long terminal repeats
  • a "tissue-specific promoter" is employed in the gene delivery vehicles of the invention and refers to transcriptional promoter/enhancer or locus defining elements, or other elements which control gene expression as discussed above, which are preferentially active in a limited number of tissue types.
  • tissue-specific promoters include the PEPCK promoter, HER2/neu promoter, casein promoter, IgG promoter, Chorionic Embryonic Antigen promoter, elastase promoter, porphobilinogen deaminase promoter, insulin promoter, growth hormone factor promoter, tyrosine hydroxylase promoter, albumin promoter, alphafetoprotein promoter, acetyl-choline receptor promoter, alcohol dehydrogenase promoter, a or b globin promoters, T-cell receptor promoter, or the osteocalcin promoter.
  • an "event-specific promoter” refers to transcriptional promoter/enhancer or locus defining elements, or other elements that control gene expression as discussed above, whose transcriptional activity is altered upon response to cellular stimuli.
  • Representative examples of such event- specific promoters include thymidine kinase or thymidilate synthase promoters, ⁇ or ⁇ interferon promoters and promoters that respond to the presence of hormones (either natural, synthetic or from other non-host organisms, e.g., insect hormones).
  • Practice of the invention includes establishing that the patient has a disease in which a population of cells in the patient express a foreign antigen.
  • This disease could be, for example cancer or a disease manifesting a population of aberrant cells, where the population is created by infection with a pathogen.
  • An example of the latter is Leishmania.
  • Cancer that is treated by the method and constructs of the invention includes, for example melanoma, lymphoma, lung cancer, and glioma. Certain patients having, for example, Hodgkin's lymphoma, or breast or ovarian endometrial cancers or pancreatic cancers, may be tested for expression of the M-CSF receptor, c-ftns, before treatment.
  • the practitioner may determine before using M-CSF ⁇ treatment that the patient's tumor cells do not express significant amounts of a functional of an M-CSF ⁇ receptor, c-fms, by use of an antibody to c-fms.
  • Cells expressing high levels of c-fms may be susceptible to autocrine or juxtacrine activation of the cancer cell upon addition of M-CSF ⁇ .
  • tumors may be effectively treated by combined therapy of M-CSF ⁇ and a prodrug, for example, thymidine kinase (TK) gene followed by treatment with a prodrug, for example gancyclovir, AZT, ddC, FIAU, FIAC or DHPG in the case of HSV TK, in order to kill all M-CSF ⁇ expressing cells if it appears that the therapy is augmenting the tumor growth rather than regressing it.
  • a prodrug for example, thymidine kinase (TK) gene
  • a prodrug for example gancyclovir, AZT, ddC, FIAU, FIAC or DHPG in the case of HSV TK
  • a prodrug for example gancyclovir, AZT, ddC, FIAU, FIAC or DHPG in the case of HSV TK
  • Tumor cells that may express c-fms include, for example, certain ovarian cancer
  • the M-CSF ⁇ gene therapy described herein can be applied to treating women having recurrent spontaneous abortions for the purpose of preventing abortions in these women.
  • Diagnosis and monitoring of a patient can include diagnosis of a cancer treatable by a therapy including administration of a mutant M-CSF ⁇ , which can be accomplished by standard cancer diagnostic procedures, and may also include a tumor biopsy and antibody test for M-CSF receptors (c-fms) on the tumor cell surfaces.
  • Leishmania, or conditions in which the body is infected with a foreign pathogen can be similarly diagnosed by standard diagnosis.
  • Subsequent monitoring of the patient can include periodic diagnostic tests following administration of the therapy.
  • a polynucleotide encoding a native M- CSF ⁇ may be administered, for example in conjunction with a soluble M-CSF, and an inhibitor of cleavage of an M-CSF ⁇ from the cell membrane.
  • administering refers to the process of delivering to a patient a therapeutic agent, or a combination of therapeutic agents.
  • the process of administration can be varied, depending on the therapeutic agents and the desired effect. For example, where several therapeutic agents are co-administered, one agent, or one combination of agents, may be delivered first, followed by a second or also a third delivery of a different therapeutic agent or several different therapeutic agents.
  • Administration can be accomplished by any means appropriate for the therapeutic agent, for example, oral means, and parenteral means, including intravenous, subcutaneous, intraarterial, intrathecal and intramuscular delivery, topical and mucosal delivery, including nasal delivery.
  • a gene therapy protocol is considered an administration including an administration of a polynucleotide that is capable of being expressed in the patient.
  • the polypeptide expressed in the patient as a result of the gene therapy protocol including an administration of a polynucleotide can be, for example, a therapeutic agent or an immunoprophylactic agent.
  • pharmaceutically acceptable carrier refers to any well known pharmaceutical carrier (e.g., physiologic saline, D5 glucose, sucrose) for the administration of a therapeutic agent, which may include, for example, a polypeptide, polynucleotide, protein, small molecule, peptoid, or peptide, that does not itself induce the production of antibodies harmful to the individual receiving the composition, and which may be administered without undue toxicity.
  • a pharmaceutical carrier e.g., physiologic saline, D5 glucose, sucrose
  • Co-administration refers to administration of one or more therapeutic agents, alone or in combination, in course of a given treatment of a patient.
  • the agents may be administered with the same pharmaceutical carrier, or different carriers. They may be administered by the same administration means, for example intramuscular injection, or different means, for example also oral administration in an enteric coated capsule, or nasal spray.
  • the agent may be the same type of agent or different types of agents, for example polynucleotides, polypeptide, or small molecules.
  • the time of administration may be exactly the same time, or one therapeutic agent may be administered before or after another agent. Thus a co-administration can be simultaneous, or consecutive. The exact protocol for a given combination of therapeutic agents it determined considering the agents and the condition being treated, among other considerations.
  • a “therapeutically effective amount” or an “effective amount” is that amount that will generate the desired therapeutic outcome. For example, if the therapeutic effect desired is a regression of a tumor, a therapeutically effective amount will be that amount that causes the tumor to regress either in whole or in part.
  • a therapeutically effective amount can be an amount administered in a dosage protocol that includes days or weeks of administration, for example. Where the therapeutic effect is an inhibition of M-CSF ⁇ convertase, the therapeutically effective amount is that amount that will cause a slower action or reduction in biological activity of an M-CSF ⁇ convertase, or an inhibition of an M-CSF ⁇ convertase catalytic activity, and thus a reduction in release of M-CSF ⁇ from a cell membrane.
  • This reduction can be, for example at least a 50% reduction of the rate of release as compared to the wild type, or to a cell releasing an M-CSF ⁇ in the absence of an inhibitor.
  • the therapeutic effect desired can also be a chemoattractant, cell proliferation, or differentiation effect, for example the effect that can be achieved on macrophages by administration of soluble M-CSF, or by administration of a polynucleotide encoding M-CSF ⁇ .
  • a therapeutic effect can be reduction of a population of diseased cells in vivo or in vitro.
  • an effective amount will depend on the variables of gene therapy that impact the effectiveness of the gene therapy, such as, for example, transformation efficiency, in vivo or ex vivo expression levels of the transformed gene, the nature of the tissue and cells being transformed, and other factors that may come into play with the particular system of gene therapy with a particular mutant or native gene or combination of genes.
  • One skilled in the art would be able to determine a "therapeutically effective amount" by beginning with a small amount and increasing the dosage until the desired therapeutic effect is achieved.
  • inhibitory amount or the term "a sufficient amount of an inhibitor” both as used herein refer to that amount that is effective for production of inhibition of a protein that has biological activity, including for example inhibition of an M-CSF ⁇ convertase, or inhibition of a biological interaction involving two or more molecules.
  • the precise inhibitory amount of an inhibitor varies depending upon the health and physical condition of the individual to be treated, the capacity of the individual's ability to adjust to the change in metabolism and body size, the formulation, and the attending physician's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.
  • a sufficient amount of an M-CSF ⁇ convertase inhibitor will be that amount that slows the release of M-CSF ⁇ from a cell membrane, and which reduce the M-CSF ⁇ convertase molecules available for cleavage of M-CSF ⁇ , for example, by reducing the effectiveness of an M-CSF ⁇ convertase.
  • Gene delivery vehicles for delivery of constructs including a coding sequence of a therapeutic of the invention, to be delivered to the mammal for expression in the mammal, for example, an M-CSF ⁇ coding sequence, or also including a nucleic acid sequence of all or a portion of an M-CSF ⁇ mutant coding sequence for delivery can be administered either locally or systemically.
  • constructs can utilize viral or nonviral vector approaches in in vivo or ex vivo modality. Expression of such coding sequence can be induced using endogenous mammalian or heterologous promoters. Expression of the coding sequence in vivo can be either constitutive or regulated.
  • the M-CSF ⁇ native or mutant coding sequence is expressed in the mammal, it can be expressed as soluble M-CSF ⁇ , or as a membrane-bound M-CSF ⁇ mutant, both or either including, for example, all of the M-CSF ⁇ coding sequence, or a biologically active portion, variant, derivative or fusion of M-CSF ⁇ .
  • in vivo administration refers to administration to a patient a polynucleotide encoding a polypeptide for expression in the patient.
  • direct in vivo administration involves transfecting a patient's tumor cell with a coding sequence without removing the tumor cell from the patient.
  • direct in vivo administration may include direct injection in the region of, for example, a tumor, of the DNA encoding the polypeptide of interest, resulting in expression in the patient's cells.
  • ex vivo administration refers to transfecting or transducing a cell, for example, a tumor cell, that is removed from the patient and that is then replaced in the patient after the transfection or transduction.
  • Ex vivo administration can be accomplished by removing cells from a patient, optionally selecting for cells to transform (i.e., tumor cells or cells bearing a foreign antigen), rendering the selected cells incapable of replication, transforming the selected cells with a polynucleotide encoding a gene for expression (i.e., a mutant M-CSF ⁇ ), including also a regulatory region for facilitating the expression, and placing the transformed cells back into the patient for expression of the mutant M-CSF ⁇ .
  • a vector construct that directs the expression of an M- CSF ⁇ and other anti-tumor agents is directly administered to a tumor.
  • Various methods may be used within the context of the present invention to directly administer the vector construct to the tumor.
  • a small metastatic lesion is located, and the vector is injected several times in several different locations within the body of tumor.
  • arteries that serve a tumor are identified using well known techniques, and the vector injected into such an artery, in order to deliver the vector directly into the tumor.
  • a tumor that has a necrotic center may be aspirated, and the vector injected directly into the now empty center of the tumor.
  • the vector construct is directly administered to the surface of the tumor, for example, by application of a topical pharmaceutical composition (e.g., DMSO) containing the vector construct, or preferably, a recombinant retroviral vector carrying the vector construct.
  • a topical pharmaceutical composition e.g., DMSO
  • a recombinant retroviral vector carrying the vector construct e.g., X-ray imaging may be used to assist in certain of the above delivery methods.
  • Methods for inhibiting the growth of a selected tumor, comprising the step of delivering to a warm-blooded animal a vector construct which directs the expression of an M-CSF ⁇ polypeptide or, in addition, other anti-tumor agents, such that the growth of the tumor is inhibited.
  • nucleic acids which encode M-CSF ⁇ , M-CSF ⁇ mutants, or, in addition, other anti-tumor agent(s) may also be administered to a patient by a variety of methods. Representative examples include transfection by various physical methods, such as lipofection (Feigner et al, Proc. Natl. Acad. Sci.
  • Retroviral vectors may be used to generate transduction competent retroviral vector particles by introducing them into appropriate packaging cell lines.
  • An other embodiment of the invention is directed to the in vivo administration of a first gene encoding M-CSF ⁇ with a second gene encoding a prodrug activator, for example a second gene encoding a thymidine kinase (TK), preferably a Herpes simplex virus or varicella-zoster virus thymidine kinase, or E. coli guanine phosphotransferase (gpt), or cytosine deaminase (CD).
  • TK thymidine kinase
  • gpt E. coli guanine phosphotransferase
  • CD cytosine deaminase
  • the therapy including administration of M-CSF ⁇ or an M- CSF ⁇ mutant, in conjunction with a gene encoding a prodrug activator and prodrug, can be immunomodulatory.
  • An example of administration of an AAV vector encoding TK and a cytokine is described in Yoshida et al, Gene Therapy 5:957-964 (1996), incorporated by reference.
  • the gene encoding the prodrug activator is expressed from its own vector, or from the same vector as the M-CSF ⁇ or M-CSF ⁇ mutant.
  • Either vector system (a single vector, or two vectors) is administered by in vivo or ex vivo means as described herein.
  • the addition of TK or other prodrug activator facilitates further immunomodulatory effect supporting the effect achieved by M-CSF ⁇ , and in addition, addition of the prodrug can activate the killing of transfected cancer cells.
  • a chaperone molecule can be administered before, contemporaneously with or after administration of the polynucleotide therapeutic, and the chaperone molecule can be, for example, a heat shock protein, such as, for example, hsp70.
  • the polynucleotide being expressed in the patient can be linked to an inducible promoter, for example a tissue specific promoter, for the purpose of, for example, ensuring expression of the polynucleotide only in the desired target cells.
  • the polynucleotide can be flanked by nucleotide sequences suitable for integration into genome of the cells of that tissue. Viral vectors that integrate, such as retroviral vectors, are preferred.
  • administering can be made directly to the tumor tissue, for expression of the therapeutics in the tumor cells, as described in WO 94/21792, incorporated by reference in full.
  • combination therapeutic agents including genes encoding M-CSF ⁇ or an M-CSF ⁇ mutant and other anti-tumor or immunomodulatory agents are administered alone or together.
  • the co-administration can be simultaneous and achieved, for example, by placing polynucleotides encoding the agents in the same vector, or by putting the agents, whether polynucleotide, polypeptide, or other drug, in the same pharmaceutical composition, or by administering the agents in different pharmaceutical compositions injected at about the same time in the same location.
  • the second agent can be administered by direct injection as appropriate for the goals of the therapy.
  • the prodrug is administered at the same location as the prodrug activator.
  • a co-administration protocol can include a combination of administrations to achieve the goal of the therapy.
  • the co-administration can include subsequent administrations as is necessary, for example, repeat in vivo direct injection administrations of an M-CSF ⁇ or an M-CSF ⁇ mutant, repeat administrations of an M-CSF ⁇ convertase inhibitor, and repeat administrations of a soluble recombinant M- CSF.
  • Examples of co-administrations applicable to practicing the present invention, and applicable to the admimstration of M-CSF ⁇ or an M-CSF ⁇ mutant with other anti- tumor agents, are described in WO 93/06867.
  • Example specific to an administration of a prodrug activator such as TK, and a prodrug for the purpose of selectively ablating genetically altered cells is described in WO 92/05262 and U.S. Patent No. 5,691,177 (Gruber) which issued November 25, 1997 and which is expressly incorporated by reference herein.
  • a cellular response may also be generated by administration of a bacteria which expresses an anti-tumor agent such as those discussed above, on its cell surface.
  • an anti-tumor agent such as those discussed above, on its cell surface.
  • Representative examples include BCG (Stover, Nature 557:456-458, 1991) and Salmonella (Newton et al, Science 244:70-72, 1989).
  • a therapy that provides administration of an antibody of a multi- drug resistant (MDR) cancer with anti-MDR antibody can be part of a combination therapy that also includes administration of an M-CSF, or a mutant M-CSF gene.
  • MDR multi- drug resistant
  • a method for inhibiting the growth of a selected tumor in a warmblooded animal, by an ex vivo administration that includes (a) removing tumor cells associated with the selected tumor from a warm-blooded animal, (b) infecting the removed cells with a vector construct which directs the expression of M-CSF ⁇ and optionally also at least one other anti-tumor agent, and (c) delivering the infected cells to a warm-blooded animal, such that the growth of the selected tumor is inhibited by immune responses generated against the gene-modified tumor cell.
  • a single cell suspension is generated by, for example, physical disruption or proteolytic digestion.
  • division of the cells may be increased by addition of various factors such as melanocyte stimulating factor for melanomas or epidermal growth factor for breast carcinomas, in order to enhance uptake, genomic integration and expression of the recombinant viral vector.
  • the removed cells are returned to the same animal, whereas in another embodiment, the cells are utilized to inhibit the growth of selected tumor cells in another, allogeneic, animal. In such a case it is generally preferable to have histocompatibility matched animals (although not always, see, e.g., Yamamoto et al., "Efficacy of Experimental FIV Vaccines," 1st International Conference of FIV researchers, University of California at Davis, September 1991).
  • a method for inhibiting the growth of a selected tumor in a warm-blooded animal comprising the steps of (a) removing tumor cells associated with the selected tumor from a warm-blooded animal, (b) transfecting or transducing the cells with a vector construct which directs the expression of an M-CSF ⁇ polypeptide, including a mutant M-CSF ⁇ polypeptide, and optionally also another anti-tumor agent such that the cells are capable of expressing said anti-tumor agent, and (c) delivering the cells from step (b) to an allogeneic warm-blooded animal, such that the growth of the selected tumor is inhibited.
  • a variety of cells may be utilized, including for example, human, macaque, dog, rat, and mouse cells. Cells may be removed from a variety of locations including, for example, from a selected tumor.
  • a vector construct may be inserted into non-tumorigenic cells, including for example, cells from the skin (dermal fibroblasts), or from the blood (e.g., peripheral blood leukocytes). If desired, particular fractions of cells such as a T cell subset or stem cells may also be specifically removed from the blood (see, for example, PCT WO 91/16116, an application entitled "Immunoselection Device and Method").
  • Vector constructs may then be contacted with the removed cells utilizing any of the above-described techniques, followed by the return of the cells to the warm-blooded animal, preferably to or within the vicinity of a tumor.
  • the above-described methods may additionally comprise the steps of depleting fibroblasts or other non-contaminating tumor cells subsequent to removing tumor cells from a warm-blooded animal, and/or the step of inactivating the cells, for example, by irradiation.
  • a therapeutic agent can be administered to a patient for the purpose of reducing a population of diseased cells for example, a patient having a cancer treatable by administration of a native or mutant M-CSF ⁇ , or a patient having Leishmania, for example, in a protocol that includes administration of several therapeutic agents.
  • the administration of an M-CSF ⁇ polynucleotide is accomplished, for example, as described above, and then either at the same time, for example, in the same pharmaceutical composition, and for example, in the same administration, an inhibitor of cleavage of M-CSF ⁇ from the cell membrane can also be administered.
  • the soluble form of M-CSF can be administered indirectly in a polynucleotide form, for expression in the patient, or can be administered as M-CSF, for example administered parenterally, and in the case of cancer, locally at a tumor site.
  • the inhibitor of an M-CSF ⁇ cleavage event can be, for example, an inhibitor of an M-CSF ⁇ convertase, for example, a hydroxamic acid inhibitor, and as such can be a small organic molecule, a peptide, a peptoid, a ribozyme, an antisense molecule, or a polypeptide, or a coding polynucleotide.
  • a polynucleotide mutant M-CSF ⁇ can be admimstered first in a viral vector, followed by an injection of a small molecule inhibitor of an M-CSF ⁇ cleavage event at about 12 to 15 hours after the initial administration of the M-CSF ⁇ polynucleotide, followed by parenteral admimstration of a soluble M-CSF polypeptide at about 48 hours after the administration of the M-CSF ⁇ .
  • M-CSF ⁇ polynucleotide with one or both of a soluble M-CSF and an inhibitor of cleavage of M-CSF ⁇ from the cell membrane.
  • the most efficacious administration protocol for a given disorder can be determined by reference to preclinical studies in animal tumor models, and may also be fine-tuned by one skilled in the art on patients in clinical trials. It is contemplated that repeat administrations of one or all of a therapeutic agent in a combination administration are required to further the efficacy of the therapy.
  • a soluble form of M-CSF may have to be supplied in a co-administration with the mutant M-CSF ⁇ , for example, in a parenteral administration of bioactive soluble M-CSF protein or a pegylated form of such protein, or in an additional administration of a polynucleotide encoding an M-CSF ⁇ , to provide the macrophage attraction to the tumor cells.
  • the soluble M-CSF can be administered, for example, about 12 hours to 48 hours after the administration of the M-CSF ⁇ polynucleotide, or up to about 1 week after administration of the M-CSF ⁇ polynucleotide, depending upon the tumor type, vector, and therapy combination. Again, it may be desirable to regulate the ratio of released to membrane-bound M-CSF ⁇ to optimize the killing of different tumor types and the immune response achieved by the therapy.
  • the multiple gene delivery vehicles or combination therapeutic agents may be administered to animals or plants.
  • the animals can be a warm-blooded animals, for example, mice, chickens, cattle, pigs, pets such as cats and dogs, horses, and humans.
  • a patient suffering from a non-metastatic, but otherwise untreatable tumor such as glioblastoma, astrocytoma, or other brain tumor, is treated, for example, by injecting purified, concentrated retroviral vector directly into the tumor, the vector encoding HSVTK and in combination an M-CSF ⁇ mutant or wild-type M-CSF ⁇ sequence.
  • the vector is preferentially integrated and expressed in tumor cells since only growing cells are transducible with retroviral vectors.
  • the vector expresses HSVTK in an unregulated fashion or, to promote greater tumor specificity, may express HSVTK from a tissue or event specific promoter that is preferentially expressed in the tumor.
  • a vector that expresses HSVTK from the CEA promoter may be utilized to treat breast or liver carcinomas.
  • Multiple injections (>10) of vector (approximately 1 ml with a titer of lxlO ⁇ -lxl ⁇ l 1 cfu) can be delivered over an extended period of time (>3 months) since the purified vector contains non-immunogenic quantities of protein ( ⁇ 1 mg protein per
  • vector may be delivered stereotactically before or after debulking surgery or chemotherapy.
  • the transduced tumor cells may be eliminated by treating the patient with pro-drugs that are activated by HSVTK, such as acyclovirs gancyclovir or AZT.
  • Metastatic, but highly localized cancers may be treated according to the methods of the present invention.
  • vector or vector producing cell lines may be injected directly into the peritoneal cavity.
  • rapidly growing tumors are preferentially transduced in vivo by a HSVTK gene delivery vehicle, and a cytokine encoding gene delivery vehicle, including a gene encoding M-CSF ⁇ , and may be subsequently destroyed by administering acyclovir or gancyclovir to the patient.
  • the cells destroyed by the drug will elicit greatly enhanced immune responses if there is a local production of cytokine, preferably M-CSF ⁇ or a mutein thereof.
  • patients with metastatic, disseminated cancer may also be treated according to the methods of the present invention.
  • primary carcinomas that have metastasized to, for example, the liver may be injected directly with viral vector or vector producing cell line of the by inserting a syringe, possibly targeted by stereotaxis, through the body wall.
  • Tumors in the lung or colon may similarly be accessed by bronchoscopy or sigmoidoscopy, respectively.
  • Tumor cells which have been transduced in vivo by, for example, a vector which expresses HSVTK may then be destroyed by administration of acyclovir or gancyclovir to the patient, giving rise to an augmented anti-tumor response in the presence of cytokines which may be present due to the second gene delivery vehicle in the combination.
  • a variety of additional therapeutic compositions may be co-administered or sequentially administered to a warm-blooded animal, in order to inhibit or destroy a pathogenic agent.
  • Such therapeutic compositions may be administered directly, or, within other embodiments, expressed from independent gene delivery vehicles.
  • a gene delivery vehicle that directs the expression of both a cytotoxic gene or gene product, and a gene which encodes the therapeutic composition (e.g., a non- vector derived gene as discussed above) may be administered to the warm-blooded animal, in order to inhibit or destroy a pathogenic agent.
  • vectors which deliver and express both the HSVTK gene and a gene coding for an immune accessory molecule, such as human M-CSF ⁇ or an M-CSF ⁇ mutant may be administered to the patient followed by or with another therapeutic vector (e.g., encoding a second cytokine, such as IL-2) or by administration of soluble M-CSF polypeptide.
  • one gene may be expressed from the vector LTR and the other may utilize an additional transcriptional promoter found between the LTRs, or may be expressed as a polycistronic mRNA, possibly utilizing an internal ribosome binding site.
  • the patient's immune system is activated due to the expression of M-CSF ⁇ and/or IL-2 and/or soluble M-CSF.
  • the overall tumor burden itself may be reduced by treating the patient with acyclovir or gancyclovir (or other appropriate purine or pyrimide-based prodrug), allowing more effective immune attack of the tumor. Infiltration of the dying tumor with inflammatory cells, in turn, increases immune presentation and further improves the patient's immune response against the tumor.
  • Any therapeutic of the invention including, for example, polynucleotides for expression in the patient, or ribozymes or antisense oligonucleotides, can be formulated into an enteric coated tablet or gel capsule according to known methods in the art. These are described in the following patents: U.S. Patent No. 4,853,230, EP 225,189, AU 9,224,296, AU 9,230,801, and WO 92144,52, which are incorporated herein by reference. Such a capsule is administered orally to be targeted to the jejunum.
  • expression of the polypeptide, or inhibition of expression by, for example a ribozyme or an antisense oligonucleotide is measured in the plasma and blood, for example by use of antibodies to the expressed or non- expressed proteins.
  • Administration of a therapeutic of the invention includes administering a therapeutically effective dose of the therapeutic, by a means considered or empirically deduced to be effective for inducing the desired, therapeutic effect in the patient. Both the dose and the administration means can be determined based on the specific qualities of the therapeutic, the condition of the patient, the progression of the disease, and other relevant factors.
  • Administration for the therapeutic agents of the invention can include, for example, local or systemic administration, including for example parenteral administration, including injection, topical administration, oral administration, catheterization, laser-created perfusion channels, a particle gun, and a pump.
  • Parenteral administration can be, for example, intravenous, subcutaneous, intradermal, or intramuscular, administration.
  • the therapeutics of the invention can be administered in a therapeutically effective dosage and amount, in the process of a therapeutically effective protocol for treatment of the patient.
  • the initial and any subsequent dosages administered will depend upon the patient's age, weight, condition, and the disease, disorder or biological condition being treated.
  • the dosage and protocol for administration will vary, and the dosage will also depend on the method of administration selected, for example, local or systemic administration.
  • the dosage is typically in the range of about 5 ⁇ g to about 50 mg/kg of patient body weight depending upon the cytokine and the health of the patient, preferably about 50 ⁇ g to about 5 mg/kg, more preferably about 100 ⁇ g to about 500 ⁇ g/kg of patient body weight.
  • Dosages for nonviral gene delivery vehicles are described for example in US 5,589,466 and US 5,580,859. Dosage of nonviral gene delivery vehicles can be 1 ⁇ g, preferably at least 5 or 10 ⁇ g, and more preferably at least 50 or 100 ⁇ g of polynucleotide, providing one or more dosages.
  • an effective DNA or mRNA dosage will be about 0.05 mg/kg to about 50 mg/kg, and usually about 0.005-5 mg/kg.
  • a formulation having the naked polynucleotide in an aqueous carrier is injected into tissue in amounts of from 10 ⁇ l per site to about 1 ml per site, and the concentration of the polynucleotide in the formulation is about 0.1 ⁇ g/ml to about 20 ug/ml.
  • vectors containing expressable constructs of coding sequences, or non-coding sequences are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a gene therapy protocol, preferably about 500 ng to about 50 mg, per injection or administration.
  • Non-coding sequences that act by a catalytic mechanism may require lower doses than non-coding sequences that are held to the restrictions of stoichometry, as in the case of, for example, antisense molecules, although expression limitations of the ribozymes may again raise the dosage requirements of ribozymes being expressed in vivo in order that they achieve efficacy in the patient.
  • Factors such as method of action and efficacy of transformation and expression are therefore considerations that will effect the dosage required for ultimate efficacy for DNA and nucleic acids.
  • microgram ( ⁇ ) amounts per kilogram of patient may be sufficient, for example, in the range of about 1 ⁇ g/kg to about 500 mg/kg of patient weight, and about 100 ⁇ g/kg to about 5 mg/kg, and about 1 ⁇ g/kg to about 50 ⁇ g/kg, and, for example, about 10 ug/kg.
  • the potency also affects the dosage, and the dosage is typically in the range of about 1 ⁇ g/kg to about 500 mg/kg of patient weight, more typically about 100 ⁇ g/kg to about 5 mg/kg, and most typically about 1 ⁇ g/kg to about 50 ⁇ g/kg.
  • the individual doses for viral gene delivery vehicles are normally used are 10 ⁇ to l ⁇ 9 c.f.u. (colony forming units of neomycin resistance titered on HT1080 cells). These are administered at one to four week intervals for three or four doses initially. If needed, subsequent booster shots are given as one or two doses after 6-12 months, and thereafter annually.
  • Dosages for AAV containing delivery systems are in the range of about 10 9 to about 10 1 x particles per body.
  • compositions comprising a recombinant viral vector as described above, in combination with a pharmaceutically acceptable carrier or diluent.
  • a pharmaceutically acceptable carrier or diluent may be prepared either as a liquid solution, or as a solid form (e.g., lyophilized) which is suspended in a solution prior to administration.
  • the composition may be prepared with suitable carriers or diluents for either surface administration, injection, oral, or rectal administration.
  • compositions are nontoxic to recipients at the dosages and concentrations employed.
  • Representative examples of carriers or diluents for injectable solutions include water, isotonic saline solutions which are preferably buffered at a physiological pH (such as phosphate- buffered saline or Tris-buffered saline), mannitol, dextrose, glycerol, and ethanol, as well as polypeptides or proteins such as human serum albumin.
  • a particularly preferred composition comprises a vector or recombinant virus in 10 mg/ml mannitol, 1 mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl.
  • the recombinant vector since the recombinant vector represents approximately 1 mg of material, it may be less than 1% of high molecular weight material, and less than 1/100,000 of the total material (including water).
  • This composition is stable at -70°C for at least six months.
  • All of the therapeutic agents that are employed in the method of the present invention can be incorporated into one or more appropriate pharmaceutical compositions that includes a pharmaceutically acceptable carrier for the agents.
  • the pharmaceutical carrier for the agents may be the same or different for each agent. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and inactive viruses in particles. Such carriers are well known to those of ordinary skill in the art.
  • Pharmaceutically acceptable salts can be used therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like
  • organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • Pharmaceutically acceptable carriers in therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • the therapeutic compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared.
  • Liposomes are included within the definition of a pharmaceutically acceptable carrier.
  • Pharmaceutical compositions are provided comprising a recombinant retrovirus or virus carrying one of the above-described vector constructs, in combination with a pharmaceutically acceptable carrier or diluent.
  • the composition may be prepared either as a liquid solution, or as a solid form (e.g., lyophilized) which is suspended in a solution prior to administration.
  • the composition may be prepared with suitable carriers or diluents for either surface administration, injection, oral, or rectal administration.
  • compositions are nontoxic to recipients at the dosages and concentrations employed.
  • Representative examples of carriers or diluents for injectable solutions include water, isotonic saline solutions which are preferably buffered at a physiological pH (such as phosphate-buffered saline or Tris-buffered saline), mannitol, dextrose, glycerol, and ethanol, as well as polypeptides or proteins such as human serum albumin.
  • a particularly preferred composition comprises a vector or recombinant virus in 10 mg/ml mannitol, 1 mg/ml HSA, 20 mM Tris, pH 7.2, and 150 mM NaCl.
  • the recombinant vector since the recombinant vector represents approximately 1 g of material, it may be less than 1% of high molecular weight material, and less than 1/100,000 of the total material (including water). This composition is stable at -70°C for at least six months.
  • the pharmaceutically acceptable carrier or diluent may be combined with the gene delivery vehicles to provide a composition either as a liquid solution, or as a solid form (e.g., lyophilized) which can be resuspended in a solution prior to administration.
  • the two or more gene delivery vehicles can be administered via traditional direct routes, such as buccal/sublingual, rectal, oral, nasal, topical, (such as transdermal and ophthalmic), vaginal, pulmonary, intraarterial, intramuscular, intraperitoneal, subcutaneous, intraocular, intranasal or intravenous, or indirectly.
  • Nonparenteral routes of administration are also contemplated by the invention. Further objects, features, and advantages of the present invention will become apparent from the detailed description.
  • Example 1 In Vivo Transduction of CT26 Tumor Cells by TK-3 This experiment was designed to demonstrate the ability of a TK-3 vector containing a gene encoding the HSVTK gene to target cells in vivo and inhibit tumor growth in the presence of gancyclovir. Firstly, the effect of the genes alone were examined, starting with TK-3. Six groups of 10 mice were injected S.C. with 1.0 x 10 ⁇
  • CT26 tumor cells CT26 tumor cells.
  • two groups of mice were injected S.C. with 1.0 x 10 ⁇ CT26TK neo cells as a control.
  • the area of the S.C. injection was circled with a water- resistant marker.
  • Twenty-four hours after tumor implantation TK-3 or ⁇ -g ⁇ / viral supernatants (0.2 ml total) formulated with polybrene (4 ⁇ g/ml) were injected into groups 3, 4, 5, and 6 (see Table 1) within the area marked by the water-resistant marker.
  • Vector administration was continued for four consecutive days with one dose of vector per day. Each vector dose contains 2.0 x 10 ⁇ cfu/ml.
  • AAV-TK-mMCSF adeno-associated virus
  • TK herpes simplex virus thymidine kinase
  • M-CSF ⁇ human macrophage colony-stimulating factor
  • FIG. 1 A diagram of pAAV-TK-mMCSF (capable of expressing TK and M-CSF ⁇ ) is shown in FIG. 1.
  • This plasmid contains the herpes simplex virus thymidine kinase (TK) gene followed by the encephalomyocarditis virus (ECMV) internal ribosome entry site (IRES) sequence, and the human M-CSF ⁇ gene.
  • the IRES sequence is from the 5' untranslated region of ECMV and allows cap-independent translation (Morgan et al. Nucl. Acids Res. 20:1293-1299). The IRES allows both genes to be expressed by a single promoter.
  • This plasmid also contains AAV ITRs for packaging into recombinant AAV vectors.
  • This plasmid was deposited with the ATCC, having ATCC No. 98335.
  • pAAV-TK-mMCSF the PvuII fragment of pAS203 (Nahreini et al. Gene 124:257-262) containing the entire AAV genome including the inverted terminal repeats was cloned into the kanamycin resistant cloning vector phss7 (Seifert et al, Proc. Natl. Acad. Sci. 55:735-739).
  • the AAV sequences encoding rep and cap were removed by digestion with EcoRl .
  • the AAV gene encoding region was replaced with an expression cassette consisting of the human cytomegalovirus (CMV) major immediate early gene promoter/enhancer including intron A (Chapman et al, Nucl Acids Res. 79:3937-3986), followed by a polylinker for cloning gene(s) of interest, and the polyadenylation site from the bovine growth hormone gene for transcriptional termination.
  • CMV human cytomegalovirus
  • intron A Camman et al, Nucl Acids Res. 79:3937-3986
  • a polylinker for cloning gene(s) of interest followed by a polylinker for cloning gene(s) of interest, and the polyadenylation site from the bovine growth hormone gene for transcriptional termination.
  • the gene for herpes simplex virus TK, the IRES (Parks et al, J. Virol. 50:376-384.), and the mMCSF gene were cloned.
  • the TK gene fragment in our vector includes nucleotides 237 to 1484 as described in McKnight et al, Nucl. Acids Res. 5:5949-5964.
  • the mMCSF gene used in this vector was derived from the mMCSF clone pcCSF-17 (Kawasaki et al, Science 250:291-296) and includes the entire coding region of pcCSF-17 with one exception.
  • the second amino acid in the leader peptide was changed from a threonine to an alanine to allow cloning the mMCSF (M-CSF ⁇ ) gene immediately 3' of the IRES.
  • Example 3 Administration of M-CSF ⁇ and TK
  • the directly administered vector is a combination of M-CSF ⁇ and TK vector in approximately equal proportions.
  • the regression of tumors with TK alone, M-CSF ⁇ alone, and other controls are included.
  • the combination of the TK and M-CSF ⁇ vectors can give greater regression than that expected of the other control treatments.
  • the proportion and titer of TK and M-CSF vector can be varied from 1:10 to 10:1 and be 10 4 , 10 5 , 10 6 , 10 7
  • a preferred preparation of the M-CSF ⁇ vector is 3 : 1.
  • the TK and M-CSF ⁇ vector is administered on alternate days. Twenty-four hours after the last vector treatment, these mice are injected LP. twice daily (AM and PM) with gancyclovir at 62.5 mg/Kg for 8 days. Finally, the mice receive a single daily dose of gancyclovir at 62.5 mg/Kg until the end of the experiment. Tumor growth is measured over a 4 week period.
  • the same procedure can be used in treating human tumors using combinations of a prodrug vector such as TK-3 and a cytokine vector such as the one encoding M-CSF ⁇ , interferon, IL2, and others.
  • mice are treated by injecting the vector producer cell line from a PCL such as DA into, or around the tumor, or both. Varying numbers of irradiated of unirradiated vector producer cells are injected with and without a polycationic reagent to improve transduction efficiencies. Control mice are injected with diluent D17 (ATCC No. CCL 183) transduced with TK-3, M-CSF ⁇ , and a CB- ⁇ -g ⁇ / vector producing cell lines (VCL). After sufficient time for in vivo transduction, approximately 2 weeks, gancyclovir injections commence and efficacy is determined by tumor measurements and/or overall survival.
  • Example 4 Direct Administration of Vector into Humans
  • the preferred location for direct administration of a vector construct depends on the location of the tumor or tumors and the type of tumor.
  • the mutant M- CSF ⁇ , or other sequences which encode anti-tumor agents or combination of these agents are preferably introduced directly into solid tumors by direct injection of the vector. They may also be delivered to leukemias, lymphomas, melanomas, gliomas, or ascites tumors. In particular, for skin lesions such as melanomas, the vector are directly injected into or around the lesion.
  • At least 10 ⁇ cfu/per vector or vector particles are administered, with preferably more than 10 ⁇ cfu in a pharmaceutically acceptable formulation (e.g., 10 mg/ml lactose, 1 mg/ml HSA, 25 mM Tris pH 7.2 and 105 mM NaCl).
  • a pharmaceutically acceptable formulation e.g. 10 mg/ml lactose, 1 mg/ml HSA, 25 mM Tris pH 7.2 and 105 mM NaCl.
  • the effected tumor is localized by X-ray, CT scan, antibody imaging, or other methods known to those skilled in the art of tumor localization.
  • Vector injection is through the skin into internal lesions, or by adaptations of bronchoscopy (for lungs), sigmoidoscopy (for colorectal or esophageal tumors) or intra-arterial or intra-blood vessel catheter (for many types of vascularized solid tumors).
  • the injection is into or around the tumor lesion.
  • the efficiency of induction of a biological response is measured by CTL assay or by delayed type hypersensitivity (DTH) reactions to the tumor.
  • Efficacy and clinical responses is determined by measuring the tumor burden using X-ray, CT scan, antibody imaging, or other methods known to those skilled in the art of tumor localization.
  • a patient is diagnosed with brain cancer, having a glioma.
  • Glioblastoma cells are removed from the patient and rendered incapable of replication, by, for example, irradiation.
  • the patient's cells are transfected with an AAV vector capable of expressing a polynucleotide encoding a mutant M-CSF ⁇ having a deletion of amino acids 161 to 165 (SEQ ID No. 3 (cDNA) and SEQ ID No. 4 (amino acid sequence)) and a polynucleotide encoding herpes simplex virus TK.
  • the cells are re-administered to the patient, and an M-CSF ⁇ convertase inhibitor, RO 31-4724, is injected at the tumor site within 15 hours after the re-administration of the altered tumor cells.
  • Soluble M-CSF polypeptide is administered at the tumor site in a single dose just after the initial administration of the cells containing an M-CSF ⁇ polynucleotide, and also administered locally at the tumor site about 48 hours after that.
  • the entire administration protocol (not including removal and re-administration of the patient's glioma cells) is repeated within 10 days of the initial re-administration of the altered tumor cells, and at 10 day intervals for about 2 months, depending upon the response of the tumor and the patient's condition.
  • the patient is monitored by magnetic resonance imaging (MRI) for tumor regression at the end of the 2 month period, and assessments for further therapy are made at that time.
  • MRI magnetic resonance imaging
  • gancyclovir administered locally to the tumor site is prescribed where tumor regression is not evident, or where tumor progression is observed.
  • a patient is diagnosed having Leishmania.
  • An AAV vector containing M-CSF ⁇ coding sequence is prepared.
  • the vector containing the M-CSF ⁇ is deposited with the ATCC, No. 98335.
  • Additionally recombinant M-CSF polypeptide, and M-CSF ⁇ convertase inhibitor, RO 31-4724 are prepared.
  • the AAV vector having M-CSF ⁇ is administered in a pharmaceutical composition by catheter into the portal vein. After a period of time of administration of the M-CSF ⁇ , the solution entering through the catheter is replaced with a solution containing the soluble M-CSF polypeptide and the RO 31-4724, which administration is carried out for a shorter period of time.
  • a readministration is prescribed weekly for several weeks, and the patient is monitored for assessments of dosage at the subsequence administrations.
  • Human HT1080 cells are transfected with plasmid constructs expressing either wild type mMCSF (M-CSF ⁇ ) ( cDNA sequence is represented in SEQ ID No. 1 or in Kawasaki et al, Science 230:291-296, 1985); or expressing mutant mMCSF (mutant M- CSF ⁇ ) (cDNA sequence is represented in SEQ ID No. 3).
  • Mutant M-CSF ⁇ molecules are made by site-directed mutagenesis as directed in Pharmacia Kit U.S.E. Mutagenesis (Rahway, NJ) from wild-type plasmid described in Kawasaki et al, Science 250:291-296 (1985).
  • Deletion mutants that deleted one or more of the amino acids between 161-165 of the wild-type sequence are made (cDNA is represented in SEQ ID No. 3 and amino acid sequence is represented in SEQ ID No. 4).
  • Supernatants from the transfected cells are collected and subjected to Western blot analysis. Any mutant that demonstrated at least a 50% reduction in release of M-CSF ⁇ into the media when compared to wild type M-CSF ⁇ is considered significant.
  • a rabbit polyclonal antiserum raised against recombinant MCSF is used in the Western blot analysis as described in Halenbeck et al, J. Biotechnology 5:45-58 (1988).
  • Example 8 Pre-clinical Ex vivo Expression of Mutant M-CSF ⁇
  • the murine melanoma K1735 is used in an experiment to test the efficacy of the rAAV vectors to deliver TK and M-CSF ⁇ to a tumor.
  • Tumor cells are tranduced ex vivo followed by implantation into syngeneic mice.
  • the mice receive ganciclovir and tumor size is measured twice a week to look for a therapeutic effect.
  • the mice show complete ablation of the tumor, they are challenged with non-transduced K1735 tumor cells to determine if a host anti-tumor immune response is generated in addition to the TK-mediated ablation.
  • K1735 cells are infected overnight with rAAV at a MOI (multiplicity of Infection) of 10 2 to 10 5 .
  • Etoposide is added at a concentration 0.2 to 2 ⁇ M. Etoposide treatment increases the transduction efficiency of rAAV in some cell types.
  • Two days after infection with rAAV the K1735 cells are trypsinized, concentrated, and resuspended in medium at a final concentration of 1.5 X 10 cells/ml. 3 X 10 6 cells in 0.2 ml medium are then injected into the supra-scapular region of syngeneic mice.
  • mice When the tumor has reached a measurable size, the mice are treated with ganciclovir at 70 mg/kg daily for 14 days. Tumors are measured twice a week. If mice show a complete response (“cure”), they will be challenged with non-transduced K1735 tumor cells to determine whether an anti-tumor immune response is generated.
  • tumors are directly injected in vivo with the rAAV vectors.
  • MOLECULE TYPE DNA (genomic)
  • Gin Ser Leu Gin Arg Leu lie Asp Ser Gin Met Glu Thr Ser Cys Gin
  • MOLECULE TYPE DNA (genomic)
  • Gin Ser Leu Gin Arg Leu lie Asp Ser Gin Met Glu Thr Ser Cys Gin 50 55 60 He Thr Phe Glu Phe Val Asp Gin Glu Gin Leu Lys Asp Pro Val Cys 65 70 75 80

Abstract

La présente invention concerne un procédé et une composition permettant de réduire une population de cellules malades par administration d'un excipient de délivrance d'un gène, excipient susceptible d'exprimer un mutant de M-CSFα présentant une aptitude diminuée à être traité par la voie protéolytique et à être libéré à partir de la membrane d'une cellule. Cette invention concerne également une combinaison d'agents thérapeutiques contenant des excipients de délivrance d'un gène exprimant M-CSFα ou un mutant de M-CSFα en combinaison, par exemple, que ce soit avec un M-CSF soluble, un inhibiteur de M-CSFα convertase, ou un excipient de délivrance d'un gène exprimant un activateur de promédicament, tel que la thymidine kinase, à la suite de l'administration du promédicament.
PCT/US1998/004802 1997-03-04 1998-03-04 COMPOSITIONS ET UTILISATIONS DE M-CSF-alpha WO1998039449A1 (fr)

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JP53892498A JP2001517079A (ja) 1997-03-04 1998-03-04 Ｍ−ＣＳＦαの組成物および方法
AU64588/98A AU6458898A (en) 1997-03-04 1998-03-04 Compositions and use of m-csf-alpha

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