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WO1999003998A1 - Procedes et compositions servant a reduire une tumeur - Google Patents

Procedes et compositions servant a reduire une tumeur Download PDF

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Publication number
WO1999003998A1
WO1999003998A1 PCT/US1998/014770 US9814770W WO9903998A1 WO 1999003998 A1 WO1999003998 A1 WO 1999003998A1 US 9814770 W US9814770 W US 9814770W WO 9903998 A1 WO9903998 A1 WO 9903998A1
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Prior art keywords
cell
fasl
cells
polynucleotide
administration
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PCT/US1998/014770
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English (en)
Inventor
Gary J. Nabel
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The Regents Of The University Of Michigan
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Priority to AU84925/98A priority Critical patent/AU8492598A/en
Publication of WO1999003998A1 publication Critical patent/WO1999003998A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A61K38/13—Cyclosporins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1841—Transforming growth factor [TGF]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis

Definitions

  • the CD95 or Fas antigen is a cell surface receptor which transduces apoptotic signals into cells. Itoh, N. et al. (1991) Cell 66:233.
  • the physiological ligand of Fas, Fas ligand (“FasL”), is a type 2 membrane protein which can transduce this signal upon cell contact(Suda, T. et al. (1993) Cell 75:1169) or in a soluble form. Nagata, S. and Goldstein, P. (1995) Science 267:1449.
  • Fas-FasL system The primary function of the Fas-FasL system is thought to be the maintenance of homeostasis in the immune system by the clonal deletion of autoreactive lymphocytes in peripheral lymphoid tissues and the elimination of expanded lymphocyte populations (Nagata, S. and Goldstein, P. (1995) Science 267:1449; Brunner, T. et al. (1995) Nature 373:441; Dhein, J. et al. (1995) Nature 373:438; and Ju, S. et al. (1995) Nature 373:444) thus contributing to homeostasis of the immune system.
  • FasL expression in normal tissue is restricted to T lymphocytes, macrophages, the cornea, the iris, ciliary bodies, the retina and Sertoli cells (Wong-Staal, F. HIV- Advances in Research and Therapy 4:3 (1994); Griffith, T. et al. (1995) Science 270: 1189).
  • T lymphocytes macrophages
  • the cornea the iris
  • ciliary bodies the retina
  • Sertoli cells Science 270: 1189.
  • FasL has been implicated in maintenance of the immune- privileged status in the eye (Griffith, T. et al. (1995), Science 270:1189) and testis (Bellgrau, D. et al. (1995) Nature 377: 630), but its mechanism of action remains unknown.
  • This invention provides a method for inhibiting the proliferation of a suitable cell by administering to the cell's locus an effective amount of FasL. It also provides a method of promoting tumor regression through the induction of a proinflammatory response or anti-tumor inflammatory response.
  • the methods are particularly suited to inhibit cellular proliferation by induction of a localized proinflammatory response, mediated, in one embodiment, by neutrophils. In a separate embodiment, it is mediated by cells of granulocytic and/or monocytic (monocytes) origin. The methods can be practiced in vitro, ex vivo or in vivo.
  • neoplastic cell selected from the group consisting of a neoplastic cell, a benign neoplasia or a normal cell.
  • the cell may or may not express the Fas (CD95/APO) receptor.
  • the neoplastic cell is a sarcoma cell, a carcinoma cell, a leukemia cell or an adenocarcinoma cell.
  • the neoplastic cell lacks functional tumor suppressor protein, e.g., p53.
  • the neoplastic cell contains an amplified oncogene.
  • FasL can be provided to the cell by localized adrninistration of a polynucleotide encoding FasL (membrane bound) or a polynucleotide encoding soluble Fas ligand, alone or in combination with a carrier, e.g., a pharmaceutically acceptable carrier.
  • a FasL or soluble FasL protein or polypeptide can be administered, alone or in conjuction with a carrier such as a pharmaceutically acceptable carrier.
  • a drug or agent that enhances endogenous FasL activation, expression, or its signalling pathway can be provided. These agents can be administered alone or in combination with a carrier such as a pharmaceutically acceptable carrier.
  • This invention further provides a method for identifying agents which modulate FasL stimulation of a localized proinflammatory response by first contacting a target cell and a control cell with a candidate therapeutic agent and assaying for induction of the proinflammatory response. One then compares the test cell assay to the control cell to determine if the agent modulates stimulation of a localized proinflammatory response.
  • Renca and CT26 cells (1X10 6 each), were stained with anti-mouse Fas antibody followed by FITC-conjugated second antibody and analyzed with FACS (upper panel). Sensitivity of Renca, but not CT26, cells to FasL exposure as determined by a chromium release assay in vitro is shown (lower panel). Renca and CT26 target cells were labeled with 5l Cr and mixed with CT26-FasL (closed circle) or CT26-neo (open circle) effector cells at various ratios. FasL-induced cytotoxicity for Renca cells was neutralized by adding 5 ⁇ g of Fas-Fc fustion protein as described in Arai, H. et al. (1997), Supra, in the medium (closed triangles). The cytotoxicity caused by FasL was measured by 51 Cr release after 4 hours of co-incubation.
  • Figure 1 B shows FasL expression after adenoviral gene transfer in Renca and CT26 cells in vitro. Renca and CT26 cells were infected with ADV-FasL at multiplicity of infection (MOI) of lxlO 3 particle/cell. At 24 and 48 hours post-transfection, the cells were harvested and the number of surviving cells were counted by Trypan-blue staining. The shown data represent the average of three wells +/- SD, and the percentage cell survival was defined as the relative number of viable cells compared to controls (circle).
  • MOI multiplicity of infection
  • FasL was detected 24 hours after infection with a Fas-Fc fusion protein (unfilled curve) compared to a control superntant (filled circle) followed by FITC-conjugated anti-IgGFc second antibody and analyzed by FACS.
  • Figure 1C shows apoptosis of cells after ADV-FasL infection in Renca but not in
  • Figure ID shows a time-course study of viability after ADV-FasL infection. Renca or CT26 cells were infected with ADV- ⁇ E1 (triangles) or ADV-FasL (squares) at an MOI of lxl 0 3 . At 24 and 48 hours post-transfection, the cells were harvested and the number of surviving cells were counted by Trypan-blue staining. The shown data represent the average of three wells +/- SD, and the precentage cells survival was defined as the relative number of viable cells compared to controls (circle).
  • Figures 2A and 2B show FasL gene transfer in Renca tumors induces apoptosis in vivo.
  • Figures 2 A and 2B show FasL gene transfer in Renca tumors induces apoptosis in vivo.
  • Renca cells (2xl0 6 ) were inoculated into Balb/C mice. After the tumor (-0.5 cm) was established, nodules were directly injected on days 7 and 8 (arrows). All adenoviral vectors were concentrated by cesium chloride density gradient centrifugation (lxlO 12 particles/ml), and 50 ⁇ l of the viral solution was injected with a 26-gauge hypodermic needle. The size of the tumor was measured in two perpendicular dimensions using calipers.
  • Figure 2B shows a histological comparison of Renca tumors treated with ADV- ⁇ E1 or ADV-FasL.
  • Hematoxylin and eosin (H&E)-stained paraffin section shows Renca cells treated with ADV- ⁇ E1 growing without any immune or inflammatory response after 24 hours. They are negative for TUNEL staining (inset).
  • Figure 3B shows FasL expression induced rejection of of CT26 cells in Balb/C recipient mice.
  • CT26-FasL cells were rejected from the immunodeficient mice.
  • Figures 4A and 4B show the histological examination of CT26-neo and CT26-FasL cells inoculated into Balb/C mice by hematoxylin and eosin staining.
  • CT26-neo cells in Balb/C mice are shown two days after inoculation; the tumor cells (T) are growing intact.
  • the CT26-FasL tumor was dissected on day 2 and stained with (i) an isotype control IgG or (ii) anti-Ly-6G antibody (RB6-8C5) specific for neutrophils and activated monocytes/macrophages (17). A large percentage of the infiltrating cells in the CT26-FasL tumor were Ly-6G positive. Original magnification: 400x.
  • Figures 5 A and 5B show expression of FasL and susceptibility to FasL in human malignancies.
  • Figure 6 provides the polynucleotide and predicted amino acid sequence of rat FasL (SEQ ID NOS: 1 and 2, respectively). The numbers above and below each line refer to the nucleotide position and amino acid positions, respectively. The putative transmembrane domain is underlined and four potential N-linked glycosylation sites (N-X- S/T) are indicated by asterisks.
  • Figure 7 provides the polynucleotide and amino acid sequence of human FasL (SEQ ID NOS: 5 and 6) as described in WO 95/18819.
  • the human FasL protein comprises an N-terminal cytoplasmic domain (amino acids 1-80), a transmembrane region (amino acids 81-105), and an extracellular domain (amino acids 106-281).
  • the extracellular domain contains the receptor binding region.
  • Soluble FasL polypeptides comprise all or part of the extracellular domain of a FasL protein, but lack the transmembrane region that would cause retention of the polypeptide on a cell membrane.
  • a heterologous signal peptide can be fused to the N-terminus such that the soluble FasL is secreted upon expression.
  • Soluble FasL can also include part of the transmembrane region or part of the cytoplasmic domain or other sequences, provided that the soluble FasL is capable of being secreted.
  • Methods of recombinantly producing FasL or isolating soluble FasL from the supernatant of cells are described in Tanaka, et al. (1995) EMBO J. 14(6): 1 129.
  • Figure 8 provides the amino acid sequences of several proteins having FasL activity for use in the methods of this invention as provided below (SEQ ID NOS: 7 to 10).
  • FasL inhibits tumor cell growth in vivo and promotes localized proinflammatory response mediated in part by neutrophils and granulocytes. Although such inhibition is expected in FasL + tumor cell lines, marked regression was unexpectedly observed after FasL gene transfer into a carcinoma in vivo, which did not express FasL. Infection by a vector encoding FasL rapidly eliminated tumor masses in the FasL + Renca tumor by inducing cell death, whereas the elimination of FasL " carcinoma cells was mediated by inflammatory cells. No pathologic abnormalities were seen in normal tissues or organs, including liver.
  • this invention provides a method of inhibiting the growth and proliferation of cells, and in particular neoplastic cells, by inducing a localized inflammatory response by introducing into the cell locus a polynucleotide coding for FasL or the FasLgene product itself.
  • Also encompassed by this invention is a method of inducing a inflammatory response by administering an effective amount of a drug or agent that activates or enhances expression of endogeneous FasL or an agent that stimulates the FasL pathway.
  • This method is useful to inhibit the proliferation cells infected by a pathogen, such as a virus or bacteria.
  • a pathogen such as a virus or bacteria.
  • This invention further provides a method for identifying agents which modulate localized FasL stimulation of a proinflammatory response by contacting a target cell and a control cell with a candidate therapeutic agent and then by assaying the target cell for localized proinflammatory response. The target cell is then compared to the response in the control cell to determine if the agent modulates localized FasL stimulation of a proinflammatory response.
  • the assaying comprises assaying for inhibition of cellular proliferation in the presence and in the absence of neutrophils.
  • the target cell is a neoplastic cell and the assaying of inhibition of cellular proliferation is by assaying for cell death or apoptosis in the presence or absence of neutrophils.
  • a cell includes a plurality of cells, including mixtures.
  • proteins proteins
  • peptides and “polypeptides” are used interchangeably and are intended to include molecules containing amino acids linearly coupled through peptide bonds.
  • the amino acids can be in the L or D form so long as the biological activity of the polypeptide is maintained.
  • the protein can be altered so as to be secreted from the cell for recombinant production and purification.
  • proteins which are post-translationally modified by reactions that include glycosylation, acetylation and phosphorylation.
  • polypeptides also include analogs, alleles and allelic variants which can contain amino acid derivatives or non-amino acid moieties that do not affect the biological or functional activity of the protein as compared to wild-type or naturally occurring protein.
  • amino acid refers both to the naturally occurring amino acids and their derivatives, such as TyrMe and PheCl, as well as other moieties characterized by the presence of both an available carboxyl group and an amine group.
  • Non-amino acid moieties which can be contained in such polypeptides include, for example, amino acid mimicking structures. Mimicking structures are those structures which exhibit substantially the same spatial arrangement of functional groups as amino acids but do not necessarily have both the ⁇ -amino and ⁇ -carboxyl groups characteristic of amino acids.
  • Mimicking structures are those structures which exhibit substantially the same spatial arrangement of functional groups as amino acids but do not necessarily have both the ⁇ -amino and ⁇ -carboxyl groups characteristic of amino acids.
  • Meins are proteins or polypeptides which have minor changes in amino acid sequence caused, for example, site-specific mutagenesis or other manipulations; by errors in transcription or translation; or which are prepared synthetically by rational design.
  • the term "peptide bond” or “peptide linkage” refers to
  • hydrophobic is intended to include those amino acids, amino acid derivatives, amino acid mimics and chemical moieties which are non-polar. Hydrophobic amino acids include Phe, Val, Trp, He and Leu.
  • positively charged amino acid refers to those amino acids, amino acid derivatives, amino acid mimics and chemical moieties which are positively charged. Positively charged amino acids include, for example, Lys, Arg and His.
  • Native polypeptides, proteins, or nucleic acid molecules refer to those recovered from a source occurring in nature or "wild-type".
  • composition is intended to mean a combination of active agent and another compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant.
  • a “pharmaceutical composition” is intended to include the combination of an active agent with a carrier, inert or active, making the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.
  • the term "pharmaceutically acceptable carrier” encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • the compositions also can include stabilizers and preservatives.
  • stabilizers and adjuvants see Martin, REMINGTON'S PHARM. SCI., 15th Ed. (Mack Publ. Co., Easton (1975)).
  • polynucleotide means single and double stranded DNA, cDNA, genome-derived DNA, and RNA, as well as the positive and negative strand of the nucleic acid that are complements of each other, including anti-sense RNA.
  • a "nucleic acid molecule” is a term used interchangeably with “polynucleotide” and each refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides, or analogs thereof. It also includes known types of modifications, for example labels which are known in the art (e.g., Sambrook, et al.
  • methylation substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl carbamate, etc.), those containing pendant moieties, such as for example, proteins (including , e.g., nuclease, toxins, antibodies, signal peptides, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylators, those with modified linkages (e.g., alpha anomeric polynucleotide, etc.), as well as unmodified forms of the polynucleotide.
  • internucleotide modifications such as, for example, those with uncharged linkages (e.g., methyl carbamate, etc.), those containing pendant moie
  • the polynucleotide can be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases.
  • the nucleotides may be complementary to the mRNA encoding the polypeptides. These complementary nucleotides include, but are not limited to, nucleotides capable of forming triple helices and antisense nucleotides.
  • Recombinant polynucleotides comprising sequences otherwise not naturally occurring are also provided by this invention, as are alterations of wild type polypeptide sequences, including but not limited to, those due to deletion, insertion, substitution of one or more nucleotides or by fusion to other polynucleotide sequences.
  • a polynucleotide is said to "encode" a polypeptide if, in its native state or when manipulated by methods well-known to those skilled in the art, it can be transcribed and/or translated to produce a polypeptide or mature protein.
  • the term polynucleotide shall include, in addition to coding sequences, processing sequences and other sequences that do not code for amino acids of the mature protein.
  • the anti-sense strand of such a polynucleotide is also said to encode the sequence.
  • polynucleotide or DNA refers to a polynucleotide that is made by the combination of two otherwise separated segments of sequence accomplished by the artificial manipulation of isolated segments of DNA by genetic engineering techniques or by chemical synthesis. In so doing one may join together DNA segments of desired functions to generate a desired combination of functions.
  • an "analog" of DNA, RNA or a polynucleotide refers to a macromolecule resembling naturally occurring polynucleotides in form and/or function (particularly in the ability to engage in sequence-specific hydrogen bonding to base pairs on a complementary polynucleotide sequence) but which differs from DNA or RNA in, for example, the possession of an unusual or non-natural base or an altered backbone. See for example,
  • a "gene” is a hereditary unit that, in the classical sense, occupies a specific position (locus) within the genome or chromosome; a unit that has one or more specific effects upon the phenotype of the organism; a unit that can mutate to various allelic forms; a unit that recombines with other such units.
  • structural genes that are transcribed into mRNAs, which are then translated into polypeptide chains (2) structural genes that are transcribed into rRNA or tRNA molecules which are used directly, and (3) regulatory genes that are not transcribed, but serve as recognition sites for enzymes and other proteins involved in DNA replication and transcription.
  • a “probe” is any biochemical labeled with radioactive isotopes or tagged in other ways for ease in identification.
  • a probe is used to identify or isolate a gene, a gene product, or a protein.
  • probes include, but are not limited to, a radioactive mRNA hybridizing with a single strand of its DNA gene, a DNA or cDNA hybridizing with its complementary region in a chromosome, or a monoclonal antibody combining with a specific protein.
  • “Hybridization” refers to hybridization reactions can be performed under conditions of different "stringency”. Conditions that increase the stringency of a hybridization reaction are widely known and published in the art: see, for example, Sambrook, et al. Supra .
  • Examples of relevant conditions include (in order of increasing stringency): incubation temperatures of 25 ° C, 37 ° C, 50 ° C, and 68 ° C; buffer concentrations of 10 x SSC, 6 x SSC, 1 x SSC, 0.1 x SSC (where SSC is 0.15 M NaCl and 15 mM citrate buffer) and their equivalent using other buffer systems; formamide concentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutes to 24 hours and washes of increasing duration, increasing frequency, or decreasing buffer concentrations.
  • Hybridization reactions can be performed under conditions of different "stringency”.
  • a low stringency hybridization reaction is carried out at about 40 °C in 10 x SSC or a solution of equivalent ionic strength/temperature.
  • a moderate stringency hybridization is typically performed at about 50 °C in 6 x SSC, and a high stringency hybridization reaction is generally performed at about 60 °C in 1 x SSC.
  • a “stable duplex" of polynucleotides, or a “stable complex” formed between any two or more components in a biochemical reaction refers to a duplex or complex that is sufficiently long-lasting to persist between the formation of the duplex or complex, and its subsequent detection.
  • the duplex or complex must be able to withstand whatever conditions exist or are introduced between the moment of formation and the moment of detection, these conditions being a function of the assay or reaction which is being performed.
  • Intervening conditions which may optionally be present and which may dislodge a duplex or complex include washing, heating, adding additional solutes or solvents to the reaction mixture (such as denaturants), and competing with additional reacting species.
  • Stable duplexes or complexes may be irreversible or reversible, but must meet the other requirements of this definition.
  • a transient complex may form in a reaction mixture, but it does not constitute a stable complex if it dissociates spontaneously or as a result of a newly imposed condition or manipulation introduced before detection.
  • stable duplexes form in an antiparallel configuration between two single-stranded polynucleotides, particularly under conditions of high stringency, the strands are essentially "complementary".
  • a double-stranded polynucleotide can be "complementary" to another polynucleotide, if a stable duplex can form between one of the strands of the first polynucleotide and the second.
  • a complementary sequence predicted from the sequence of a single stranded polynucleotide is the optimum sequence of standard nucleotides expected to form hydrogen bonding with the single-stranded polynucleotide according to generally accepted base-pairing rules.
  • a “sense” strand and an “antisense” strand when used in the same context refer to single-stranded polynucleotides which are complementary to each other. They may be opposing strands of a double-stranded polynucleotide, or one strand may be predicted from the other according to generally accepted base-pairing rules. Unless otherwise specified or implied, the assignment of one or the other strand as “sense” or “antisense” is arbitrary.
  • a linear sequence of nucleotides is "identical" to another linear sequence, if the order of nucleotides in each sequence is the same, and occurs without substitution, deletion, or material substitution. It is understood that purine and pyrimidine nitrogenous bases with similar structures can be functionally equivalent in terms of Watson-Crick base-pairing; and the inter-substitution of like nitrogenous bases, particularly uracil and thymine, or the modification of nitrogenous bases, such as by methylation, does not constitute a material substitution.
  • RNA and a DNA polynucleotide have identical sequences when the sequence for the RNA reflects the order of nitrogenous bases in the polyribonucleotide, the sequence for the DNA reflects the order of nitrogenous bases in the polydeoxyribonucleotide, and the two sequences satisfy the other requirements of this definition.
  • the sequences are a degenerate oligonucleotide comprising an ambiguous residue
  • the two sequences are identical if at least one of the alternative forms of the degenerate oligonucleotide is identical to the sequence with which it is being compared.
  • AYAAA is identical to ATAAA, if AYAAA is a mixture of ATAAA and ACAAA.
  • the sequences are identical if one strand of the first polynucleotide is identical with one strand of the second polynucleotide.
  • a polynucleotide probe is described as identical to its target, it is understood that it is the complementary strand of the target that participates in the hybridization reaction between the probe and the target.
  • a linear sequence of nucleotides is "essentially identical” or the "equivalent” to another linear sequence, if both sequences are capable of hybridizing to form duplexes with the same complementary polynucleotide. It should be understood, although not always explicitly stated that when Applicants refer to a specific polynucleotide, its equivalents are also intended. Sequences that hybridize under conditions of high stringency are more preferred. It is understood that hybridization reactions can accommodate insertions, deletions, and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align. Sequences that correspond or align more closely to the invention disclosed herein are comparably more preferred.
  • a polynucleotide region of about 25 residues is essentially identical to another region, if the sequences are at least about 80%> identical; more preferably, they are at least about 90%) identical; more preferably, they are at least about 95%) identical; still more preferably, the sequences are 100% identical.
  • a polynucleotide region of 40 residues or more will be essentially identical to another region, after alignment of homologous portions if the sequences are at least about 75%) identical; more preferably, they are at least about 80%> identical; more preferably, they are at least about 85%) identical; even more preferably, they are at least about 90%) identical; still more preferably, the sequences are 100%) identical.
  • polynucleotide sequences are essentially identical, a sequence that preserves the functionality of the polynucleotide with which it is being compared is particularly preferred. Functionality can be determined by different parameters. For example, if the polynucleotide is to be used in reactions that involve hybridizing with another polynucleotide, then preferred sequences are those which hybridize to the same target under similar conditions.
  • the T m of a DNA duplex decreases by about 10 °C for every 1%) decrease in sequence identity for duplexes of 200 or more residues; or by about 50 °C for duplexes of less than 40 residues, depending on the position of the mismatched residues (see, e.g., Meinkoth et al.).
  • Essentially identical or equivalent sequences of about 100 residues will generally form a stable duplex with each other's respective complementary sequence at about 20 ° C less than T m ; preferably, they will form a stable duplex at about 15 ° C less; more preferably, they will form a stable duplex at about 10 ° C less; even more preferably, they will form a stable duplex at about 5 °C less; still more preferably, they will form a stable duplex at about T m .
  • preferred sequences are those which encode identical or essentially identical polypeptides.
  • nucleotide differences which cause a conservative amino acid substitution are preferred over those which cause a non-conservative substitution
  • nucleotide differences which do not alter the amino acid sequence are more preferred, while identical nucleotides are even more preferred.
  • Insertions or deletions in the polynucleotide that result in insertions or deletions in the polypeptide are preferred over those that result in the down-stream coding region being rendered out of phase; polynucleotide sequences comprising no insertions or deletions are even more preferred.
  • the relative importance of hybridization properties and the encoded polypeptide sequence of a polynucleotide depends on the application of the invention.
  • a polynucleotide has the same characteristics or is the equivalent of another polynucleotide if both are capable of forming a stable duplex with a particular third polynucleotide under similar conditions of maximal stringency.
  • the polynucleotides also encode essentially identical polypeptides.
  • Constant residues of a polynucleotide sequence are those residues which occur unaltered in the same position of two or more related sequences being compared. Residues that are relatively conserved are those that are conserved amongst more related sequences than residues appearing elsewhere in the sequences.
  • “Related” polynucleotides are polynucleotides that share a significant proportion of identical residues.
  • a "degenerate" oligonucleotide sequence is a designed sequence derived from at least two related originating polynucleotide sequences as follows: the residues that are conserved in the originating sequences are preserved in the degenerate sequence, while residues that are not conserved in the originating sequences may be provided as several alternatives in the degenerate sequence.
  • the degenerate sequence AYASA may be designed from originating sequences ATACA and ACAGA, where Y is C or T and S is C or G. Y and S are examples of "ambiguous" residues.
  • a degenerate segment is a segment of a polynucleotide containing a degenerate sequence. It is understood that a synthetic oligonucleotide comprising a degenerate sequence is actually a mixture of closely related oligonucleotides sharing an identical sequence, except at the ambiguous positions. Such an oligonucleotide is usually synthesized as a mixture of all possible combinations of nucleotides at the ambiguous positions. Each of the oligonucleotides in the mixture is referred to as an "alternative form".
  • a polynucleotide "fragment” or “insert” as used herein generally represents a sub-region of the full-length form, but the entire full-length polynucleotide may also be included.
  • RNA corresponds to the gene from which it is transcribed.
  • cDNA corresponds to the RNA from which it has been produced, such as by a reverse transcription reaction, or by chemical synthesis of a DNA based upon knowledge of the RNA sequence.
  • cDNA also corresponds to the gene that encodes the RNA.
  • Polynucleotides also "correspond" to each other if they serve a similar function, such as encoding a related polypeptide, in different species, strains or variants that are being compared.
  • a "probe” when used in the context of polynucleotide manipulation refers to an oligonucleotide which is provided as a reagent to detect a target potentially present in a sample of interest by hybridizing with the target.
  • a probe will comprise a label or a means by which a label can be attached, either before or subsequent to the hybridization reaction.
  • Suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes.
  • a “primer” is an oligonucleotide, generally with a free 3' -OH group, that binds to a target potentially present in a sample of interest by hybridizing with the target, and thereafter promotes polymerization of a polynucleotide complementary to the target.
  • Processes of producing replicate copies of the same polynucleotide, such as PCR or gene cloning, are collectively referred to herein as "amplification” or "replication”.
  • amplification or “replication”.
  • RNA may be replicated, for example, by an RNA-directed RNA polymerase, or by reverse-transcribing the DNA and then performing a PCR. In the latter case, the amplified copy of the RNA is a DNA with the identical sequence.
  • PCR polymerase chain reaction
  • PCR is a reaction in which replicate copies are made of a target polynucleotide using one or more primers, and a catalyst of polymerization, such as a reverse transcriptase or a DNA polymerase, and particularly a thermally stable polymerase enzyme.
  • a PCR involves reiteratively forming three steps: "annealing”, in which the temperature is adjusted such that oligonucletide primers are permitted to form a duplex with the polynucleotide to be amplified; “elongating”, in which the temperature is adjusted such that oligonucleotides that have formed a duplex are elongated with a DNA polymerase, using the polynucleotide to which are formed the duplex as a template; and “melting”, in which the temperature is adjusted such that the polynucleotide and elongated oligonucleotides dissociate. The cycle is then repeated until the desired amount of amplified polynucleotide is obtained.
  • Methods for PCR are taught in U.S. Patent Nos. 4,683,195 (Mullis) and 4,683,202 (Mullis et al.).
  • Elements within a gene include but are not limited to promoter regions, enhancer regions, repressor binding regions, transcription initiation sites, ribosome binding sites, translation initiation sites, protein encoding regions, introns and exons, and termination sites for transcription and translation.
  • operatively linked means that the DNA molecule is positioned relative to the necessary regulation sequences, e.g., a promoter or enhancer, such that a promoter will direct transcription of RNA off the DNA molecule in a stable or transient manner.
  • Regulatory elements include but are not limited to, promoter regions, enhancer regions, repressor binding regions, transcription initiation sites, ribosome binding sites, translation initiation sites, protein encoding regions, introns and exons, and termination sites for transcription and translation.
  • a "subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.
  • a “control” is an alternative subject or sample used in an experiment for comparison purpose. A control can be "positive” or "negative”.
  • a positive control a subject or a sample from a subject, carrying such alteration and exhibiting syndromes characteristic of that disease
  • a negative control a subject or a sample from a subject lacking the altered expression and clinical syndrome of that disease
  • a "gene delivery vehicle” is defined as any molecule that can carry inserted polynucleotides into a host cell.
  • gene delivery vehicles examples include liposomes, viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmid, plasmid, fungal vectors and other recombination vehicles typically used in the art which have been described for expression in a variety of eukaryotic and prokaryotic hosts, and may be used for gene therapy as well as for simple protein expression.
  • a "viral vector” is defined as a recombinantly produced virus or viral particle that comprises a polynucleotide to be delivered into a host cell, either in vivo, ex vivo or in vitro.
  • viral vectors include retroviral vectors, adenovirus vectors, adeno- associated virus vectors and the like.
  • a vector construct refers to the polynucleotide comprising the retroviral genome or part thereof, and a transgene or polypeptide.
  • retroviral mediated gene transfer or “retroviral transduction” carries the same meaning and refers to the process by which a gene or polynucleotide sequences are stably transferred into the host cell by virtue of the virus entering the cell and integrating its genome into the host cell genome.
  • the virus can enter the host cell via its normal mechanism of infection or be modified such that it binds to a different host cell surface receptor or ligand to enter the cell.
  • retroviral vector refers to a viral particle capable of introducing exogenous nucleic acid into a cell through a viral or viral-like entry mechanism. Retro viruses carry their genetic information in the form of RNA; however, once the virus infects a cell, the RNA is reverse-transcribed into the DNA form which integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.
  • a vector construct refers to the polynucleotide comprising the viral genome or part thereof, and a transgene or polypeptide.
  • Ads adenoviruses
  • Ads are a relatively well characterized, homogenous group of viruses, including over 50 serotypes. (see, e.g., WO 95/27071) Ads are easy to grow and do not require integration into the host cell genome.
  • Ad-derived vectors particularly those that reduce the potential for recombination and generation of wild-type virus, have also been constructed, (see, WO 95/00655; WO 95/11984). Wild-type AAV has high infectivity and specificity integrating into the host cells genome. (Hermonat and Muzyczka (1984) PNAS USA 81:6466-6470; Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996).
  • Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operatively linked are well known in the art. Such vectors are capable of transcribing RNA in vitro or in vivo, and are commercially available from sources such as
  • Gene delivery vehicles also include several non-viral vectors, including DNA/liposome complexes, and targeted viral protein DNA complexes. Liposomes that also comprise a targeting antibody or fragment thereof can be used in the methods of this invention.
  • the nucleic acid or proteins of this invention can be conjugated to antibodies or binding fragments thereof which bind cell surface antigens.
  • Polynucleotides are inserted into vector genomes using methods well known in the art. For example, insert and vector DNA can be contacted, under suitable conditions, with a restriction enzyme to create complementary ends on each molecule that can pair with each other and be joined together with a ligase.
  • synthetic nucleic acid linkers can be ligated to the termini of restricted polynucleotide.
  • oligonucleotide containing a termination codon and an appropriate restriction site can be ligated for insertion into a vector containing, for example, some or all of the following: a selectable marker gene, such as the neomycin gene for selection of stable or transient transfectants in mammalian cells; enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription; transcription termination and RNA processing signals from SV40 for mRNA stability; SV40 polyoma origins of replication and ColEl for proper episomal replication; versatile multiple cloning sites; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA.
  • a selectable marker gene such as the neomycin gene for selection of stable or transient transfectants in mammalian cells
  • enhancer/promoter sequences from the immediate early gene of human CMV for high levels of transcription
  • transcription termination and RNA processing signals from SV40 for mRNA stability transcription termination and RNA processing signals
  • “Host cell” is intended to include any individual cell or cell culture which can be or have been recipients for vectors or the incorporation of exogenous polynucleotides, polypeptides and/or proteins. It also is intended to include progeny of a single cell, and the progeny may not necessarily be completely identical (in mo ⁇ hology or in genomic or total DNA complement) to the original parent cell due to natural, accidental, or deliberate mutation.
  • the cells may be procaryotic or eucaryotic, and include but are not limited to bacterial cells, yeast cells, plant cells, insect cells, animal cells, and mammalian cells, e.g., murine, rat, simian or human.
  • expression refers to the process by which polynucleotides are transcribed into mRNA and translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA, if an appropriate eucaryotic host is selected. Regulatory elements required for expression include promoter sequences to bind RNA polymerase and transcription initiation sequences for ribosome binding.
  • a bacterial expression vector includes a promoter such as the lac promoter and for transcription initiation the Shine-
  • an eucaryotic expression vector includes a heterologous or homologous promoter for RNA polymerase II, a downstream polyadenylation signal, the start codon AUG, and a termination codon for detachment of the ribosome.
  • RNA polymerase II a heterologous or homologous promoter for RNA polymerase II
  • a downstream polyadenylation signal for RNA polymerase II
  • the start codon AUG a downstream polyadenylation signal
  • a termination codon for detachment of the ribosome.
  • purified or isolated shall mean not in the native or endogenous environment. In one embodiment, it shall mean removed from constituents normally associated with the nucleic acid, vector, cell, protein or polypeptide, in its native or naturally occurring environment.
  • the polynucleotides and/or proteins may be used in purified form when in the naturally occurring form. However, the polynucleotides and/or proteins need not be utilized in the "isolated” or “purified” state when present in an unnatural form, e.g., produced from a bacterial cell.
  • Solid phase support is not limited to a specific type of support. Rather a large number of supports are available and are known to one of ordinary skill in the art. Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels. A suitable solid phase support may be selected on the basis of desired end use and suitability for various synthetic protocols.
  • solid phase support may refer to resins such as polystyrene (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE ® resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel ® , Rapp
  • an “antibody” is an immunoglobulin molecule capable of binding an antigen. As used herein, the term encompasses not only intact immunoglobulin molecules, but also anti-idiotypic antibodies, mutants, fragments, fusion proteins, humanized proteins and modifications of the immunoglobulin molecule that comprise an antigen recognition site of the required specificity.
  • An “antibody complex” is the combination of antibody (as defined above) and its binding partner or ligand.
  • an "antisense" copy of a particular polynucleotide refers to a complementary sequence that is capable of hydrogen bonding to the polynucleotide and can therefor, be capable of modulating expression of the polynucleotide. These may be DNA, RNA or analogs thereof, including analogs having altered backbones, as described above.
  • the polynucleotide to which the antisense copy binds may be in singe-stranded form or in double-stranded form.
  • This invention provides a method for stimulating an anti-tumor response or for inhibiting the proliferation of a suitable cell, by administering to the cell's locus an effective amount of FasL.
  • the method induces a localized inflammatory response, mediated, in one embodiment, by promoting lysis of the cell by neutrophils.
  • the cells express the Fas receptor and the response is mediated by death signaling or apoptosis.
  • the method can be practiced in vitro, ex vivo and in vivo. The method also is useful to develop drugs that act through the same or similar mechanism.
  • a suitable cell is a cell selected from the group consisting of any of a neoplastic cell, a benign neoplasia (e.g., nevus, or leiomyomas (uterus)) or a normal cell.
  • the cell may or may not express the Fas (CD95/APO) receptor.
  • the suitable neoplastic cell is any of a sarcoma cell, a carcinoma cell, a leukemia cell or an adenocarcinoma cell.
  • the suitable neoplastic cell lacks functional tumor suppressor protein.
  • the suitable neoplastic cell contains and/or epresses an amplified oncogene.
  • FasL can be administered by administration of an effective amount of a polynucleotide encoding membrane bound FasL or soluble Fas ligand.
  • an effective amount of FasL polynucleotide, protein or polypeptide can be administered by direct addition to the cell culture or supernatant.
  • they can be combined with a carrier, such as a pharmaceutically acceptable carrier, for in vivo use.
  • administering for in vivo and ex vivo purposes means providing the subject with an effective amount of the FasL polynucleotide or polypeptide effective to inhibit proliferation of the cell and/or induce an inflammatory response. It also includes the adminstration of an agent or drug which activates ("turns on") or enhances the expression and/or biological effect of an endogenous FasL gene.
  • Methods of administering pharmaceutical compositions in vivo are well known to those of skill in the art and include, but are not limited to, microinjection, intravenous or parenteral administration.
  • the compositions are intended for topical, oral, or local administration as well as intravenously, subcutaneously, or intramuscularly.
  • Administration can be effected continuously or intermittently throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the polynucleotide used for therapy, the vector used for therapy, the polypeptide or protein used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
  • an effective "therapeutic amount" of the composition is administered to prevent or at least partially inhibit the proliferation of the cells or to induce a localized inflammatory response.
  • the term "proinflammatory response” shall mean an inflammatory response mediated by the Fas-FasL system, which includes, but is not limited to both cellular and humoral immunity which includes inflammation mediated by neutrophils or cells of granulocytic or monocytic origin.
  • the term "neoplastic cell” is used interchangeably with the terms “cancer,” “neoplasm,” and “tumor,” and is used in either the singular or plural form, refers to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • Primary cancer cells that is, cells obtained from near the site of malignant transformation
  • a cancer cell includes not only a primary cancer cell, but any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells.
  • a "clinically detectable" tumor is one that is detectable on the basis of tumor mass; e.g., by such procedures as CAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical or immunologic findings alone may be insufficient to meet this definition.
  • FasL is intended to include, but not be limited to the polynucleotide comprising the open reading frame of the FasL polynucleotide shown in Figure 6 and 7
  • FasL is intended to include, but not be limited to the polynucleotide comprising the open reading frame of the FasL polynucleotide provided in WO 98/03648, WO 97/33617, WO
  • One of skill in the art can determine if a putative equivalent has the requisite biological activity by assaying the polynucleotide in the methods provided below. Although biological activity greater than the FasL polynucleotide provided herein is preferred, polynucleotides coding for polypeptides having less potent biological activity are useful as controls in the assays provided herein.
  • the term also is intended to include polynucleotides which correspond to the sequence shown in Figure 7, its complement, essentially identical polynucleotides or equivalent polynucleotides.
  • the term "FasL” further includes the polynucleotides coding for ligand proteins of the TNF/nerve growth factor receptor family as shown in Figure 8 (SEQ ID NOS: 8 to 15), which includes two TNF receptors (type I, ⁇ or 55kD; type II, ⁇ or 75 kD), the low-affinity nerve growth factor receptor (CD40, CD27, CD30 and OX40), biologically equivalent mutants, analogs and variants thereof.
  • polynucleotides coding for polypeptides having the sequence shown in SEQ ID NO 3, biologically equivalent mutants, analogs and variants thereof are useful as controls in the assays provided herein.
  • polynucleotide sequences that encodes biologically equivalent proteins such as amino acids as shown in Figures 6 to 8, modified by having conservative amino acid substitutions.
  • the term also includes a polynucleotide coding for soluble FasL, the polynucleotide and amino acid sequence of which is shown in Figure 7 (SEQ ID NO: 3 and 4) and disclosed in WO 95/13701, and their complements; as well as polynucleotides that hybridize to these sequences, or their complements under stringent hybridization conditions or those polynucleotides which are greater than 75%>, more preferably greater than 85%> and most preferably, greater than 90%> homologous as determined by a sequence allignment program such as such as BLAST using the default search parameters with a cutoff of a high score of 50 or above, 100 or above, or more preferably 150 or above (see http://www.ncbi.
  • polynucleotides which correspond to the sequence shown in Figure 7, its complement, an essentially identical polynucleotide or an equivalent polynucleotide. It further includes a polynucleotide sequences that encodes biologically equivalent proteins such as amino acids as shown in Figure 7 and modified by having conservative amino acid substitutions. It further includes polynucleotides coding for polypeptides having the sequence shown in SEQ ID NO 3 or WO 95/13701, biologically equivalent mutants, analogs and variants thereof. One of skill in the art can determine if a putative equivalent has the requisite biological activity by assaying the polynucleotide in the methods provided below. Although biological activity greater than the FasL polynucleotide provided herein is preferred, polynucleotide coding for polypeptides having less potent biological activity are useful as controls in the assays provided herein.
  • compositions provide a separate embodiment of the invention, which may be practiced separately or in combination with each other or another, yet undetermined composition.
  • the methods also may be combined with known anti- cancer therapies such as chemotherapy and radiation therapy for the eradication of tumors.
  • known anti- cancer therapies such as chemotherapy and radiation therapy for the eradication of tumors.
  • the methods also are useful for preventing and treating bacterial, viral and parasitic infections and may likewise be combined with known anti-infective therapies.
  • This invention further includes the polynucleotides coding for FasL and soluble FasL as defined herein in a host cell or in a gene delivery vehicle such as a liposome, a viral vector, or a plasmid.
  • the polynucleotides are operatively linked to control and expression sequences required for the expression of the polynucleotide in a cell.
  • the host cell containing the FasL polynucleotide and soluble FasL polynucleotide also is provided by this invention.
  • the host cell may be a procaryotic cell, such a bacterial cell or a eucaryotic cell such as an insect cell, a plant cell or an animal cell, such as a mammalian cell.
  • FasL also includes FasL protein and soluble FasL polypeptide, the amino acid sequences of which are provided in Figures 6 through 8, as well as analogs, muteins and variants thereof having comparable biological activity to wild-type FasL.
  • the analogs, muteins and variants thereof can be assayed in the screening method described below to determine if they have the required biological activity.
  • the method is useful when run in vitro as a quick screen or assay for anti- proliferative and/or anti-infective compounds or to assay compounds for their ability to invoke a proinflammatory response.
  • The can be used ex vivo to test agents for their biological activity prior to use in vivo. In vivo they provide therapies and can be used in combination with other known therapies.
  • the method When the method is practiced in an animal, it provides an model for anti-tumor and/or anti-infective therapies and an assay system to test additional drugs and/or therapies.
  • This invention further provides use of FasL polynucleotide and protein for the preparation of medicaments for provoking an inflammatory response and/or inhibiting the proliferation of cells.
  • any suitable gene transfer method is intended. Such methods include, but are not limited to by electroporation, transformation or transfection procedures. Sambrook et al. (1989), Supra .
  • the gene is inserted into an appropriate expression vector by methods well known in the art and as described below.
  • the object of the method has been met by noting apoptosis, necrosis or any other means of preventing cell division, reduced tumorigenicity, maturation, differentiation or reversion of the neoplastic phenotype of the cell. If the object of the method is an proinflammatory response, the chromium release assay as described below provides but one means to determine if the object of the method has been met.
  • polynucleotides encoding one or more of the embodiments of FasL can be delivered to the subject using a gene delivery vehicle.
  • the methods of this invention are intended to encompass any method of transgene transfer into and transgene expression in the subject.
  • delivery mechanisms include, but are not limited to viral mediated gene transfer, liposome mediated transfer, transformation, transfection and transduction, e.g., viral mediated gene transfer such as the use of vectors based on DNA viruses such as adenovirus, adeno-associated virus and herpes virus, as well as retroviral based vectors.
  • genetic modifications of cells in vitro, ex vivo and in vivo, employed in the present invention are accomplished by introducing a vector containing a polynucleotide as described herein.
  • a variety of different gene transfer vectors, including viral as well as non- viral systems can be used.
  • Viral vectors useful in the genetic modifications of this invention include, but are not limited to adenovirus, adeno-associated virus vectors, retroviral vectors and adeno-retroviral chimeric vectors.
  • Adenovirus and adeno-associated virus vectors useful in the genetic modifications of this invention may be produced according to methods already taught in the art. (see, e.g., Karlsson, et al. (1986) EMBO 5:2377; Carter (1992) Current Opinion in Biotechnology 3:533; Muzcyzka (1992) Current Top. Microbiol. Immunol. 158:97; GENE
  • TARGETING A PRACTICAL APPROACH (1992) ed. A. L. Joyner, Oxford University Press, NY).
  • Several different approaches are feasible. Preferred is the helper-independent replication deficient human adenovirus system.
  • adenoviral vectors based on the human adenovirus 5 are missing essential early genes from the adenoviral genome (usually
  • a transgene of interest can be cloned and expressed in cells infected with the replication deficient adenovirus.
  • adenovirus-based gene transfer does not result in integration of the transgene into the host genome (less than 0.1 % adenovirus-mediated transfections result in transgene incorporation into host DNA), and therefore is not stable, adenoviral vectors can be propagated in high titer and transfect non-replicating cells.
  • Human 293 cells which are human embryonic kidney cells transformed with adenovirus E1A/E1B genes, typify useful permissive cell lines and are commercially available from the American Type Culture Collection (ATCC). However, other cell lines which allow replication-deficient adenoviral vectors to propagate therein can be used, including HeLa cells.
  • adenovirus vectors and other viral vectors which could be used in the methods of the present invention include the following: Horwitz, M.S., Adenoviridae and Their Replication, in Fields, B. et al. (eds.) VIROLOGY, Vol. 2, Raven
  • adenovirus plasmids are also available from commercial sources, including, e.g., Microbix Biosystems of Toronto, Ontario (see, e.g., Microbix Product Information Sheet: Plasmids for Adenovirus Vector Construction, 1996). See also, the papers by Vile, et al. (1997) Nature Biotechnology 15: 840; Feng, et al.(1997)
  • Retroviral vectors useful in the methods of this invention are produced recombinantly by procedures already taught in the art.
  • WO 94/29438 describes the construction of retroviral packaging plasmids and packaging cell lines.
  • the retroviral vectors useful in the methods of this invention are capable of infecting the cells described herein.
  • the techniques used to construct vectors, and transfix and infect cells are widely practiced in the art.
  • Examples of retroviral vectors are those derived from murine, avian or primate retroviruses.
  • Retroviral vectors based on the Moloney murine leukemia virus (MoMLV) are the most commonly used because of the availability of retroviral variants that efficiently infect human cells.
  • Other suitable vectors include those based on the Gibbon Ape Leukemia Virus (GALV) or HIV.
  • GALV Gibbon Ape Leukemia Virus
  • the viral gag, pol and env sequences are removed from the virus, creating room for insertion of foreign DNA sequences. Genes encoded by the foreign DNA are usually expressed under the control of the strong viral promoter in the LTR. Such a construct can be packed into viral particles efficiently if the gag, pol and env functions are provided in trans by a packaging cell line. Thus, when the vector construct is introduced into the packaging cell, the gag-pol and env proteins produced by the cell, assemble with the vector RNA to produce infectious virions that are secreted into the culture medium.
  • MoMLV Moloney murine leukemia virus
  • the virus thus produced can infect and integrate into the DNA of the target cell, but does not produce infectious viral particles since it is lacking essential packaging sequences.
  • Most of the packaging cell lines currently in use have been transfected with separate plasmids, each containing one of the necessary coding sequences, so that multiple recombination events are necessary before a replication competent virus can be produced.
  • the packaging cell line harbors an integrated provirus.
  • the provirus has been crippled so that, although it produces all the proteins required to assemble infectious viruses, its own RNA cannot be packaged into virus. Instead, RNA produced from the recombinant virus is packaged.
  • the virus stock released from the packaging cells thus contains only recombinant virus.
  • the range of host cells that may be infected by a retrovirus or retroviral vector is determined by the viral envelope protein.
  • the recombinant virus can be used to infect virtually any other cell type recognized by the env protein provided by the packaging cell, resulting in the integration of the viral genome in the transduced cell and the stable production of the foreign gene product.
  • murine ecotropic env of MoMLV allows infection of rodent cells
  • amphotropic env allows infection of rodent, avian and some primate cells, including human cells.
  • Amphotropic packaging cell lines for use with MoMLV systems are known in the art and commercially available and include, but are not limited to, PA12 and PA317. Miller, et al. (1985) Mol. Cell. Biol. 5:431; Miller, et al. (1986) Mol. Cell. Biol. 6:2895; and Danos, et al. (1988) Proc. Natl. Acad. Set USA
  • Xenotropic vector systems exist which also allow infection of human cells.
  • the host range of retroviral vectors has been altered by substituting the env protein of the base virus with that of a second virus.
  • the resulting, "pseudotyped" virus has the host range of the virus donating the envelope protein and expressed by the packaging cell line.
  • VSV-G vesicular stomatitis virus
  • VSV-G pseudotyped vectors Since infection is not dependent on a specific receptor, VSV-G pseudotyped vectors have a broad host range.
  • the vectors will contain at least two heterologous genes or gene sequences: (i) the therapeutic gene to be transferred; and (ii) a marker gene that enables tracking of infected cells.
  • therapeutic gene can be an entire gene or only the functionally active fragment of the gene capable of compensating for the deficiency in the patient that arises from the defective endogenous gene.
  • Therapeutic gene also encompasses antisense oligonucleotides or genes useful for antisense suppression and ribozymes for ribozyme-mediated therapy.
  • a therapeutic gene may be one that neutralizes the immunosuppressive factor or counter its effects.
  • the method also can be practiced ex vivo using a modification of the method described in Lum et al. (1993) Bone Marrow Transplantation 12:565 or a modification of the method described in U.S. Patent No. 5,399,346.
  • a sample of cells such as bone marrow cells containing neoplastic cells can be removed from a subject or animal using methods well known to those of skill in the art.
  • An effective amount of FasL nucleic acid is added to the cells and the cells are cultured under conditions that favor internalization of the nucleic acid and expression of the polynucleotide by the cells.
  • the transformed cells are then returned or reintroduced to the same subject or animal (autologous) or one of the same species (allogeneic) in an effective amount and in combination with appropriate pharmaceutical compositions and carriers.
  • Non-viral vectors such as plasmid vectors useful in the genetic modifications of this invention, can be produced according to methods taught in the art. References describing the construction of non-viral vectors include the following: Ledley, F.D. (1995) Human Gene Therapy 6:1129; Miller, N. et al. (1995) FASEB Journal 9:190; Chonn, A. et al. (1995) Curr. Opin. in Biotech. 6:698; Schofield, J.P. et al. (1995) British Med. Bull. 51:56; Brigham, K.L. et al.(1993) J Liposome Res. 3:31; Brigham, K.L.
  • WO 91/06309 (16 May 1991); Feigner, P.L. et al. WO 91/17424 (14 November 1991); Solodin, et al. (1995) Biochemistry 34: 13537; WO 93/19768 (14 October 1993); Debs, et al. WO 93/25673; Feigner, P.L. et al. U.S. Patent 5,264,618 (November 23, 1993); Epand, R.M. et al. U.S. Patent 5,283,185 (February 1, 1994); Gebeyehu, et al. U.S. Patent 5,334,761 (August 2, 1994); Feigner, P.L. et al. U.S. Patent 5,459,127 (October 17, 1995); Overell, R.W. et al.
  • WO 95/28494 26 October 1995
  • Jessee WO 95/02698 (26 January 1995); Haces and Ciccarone, WO 95/17373 (29 June 1995); and Lin, et al. WO 96/01840 (25 January 1996).
  • More than one gene can be administered per vector or alternatively, more than one gene can be delivered using several compatible vectors.
  • the vector can include the regulatory and untranslated sequences.
  • the polynucleotides encoding the FasL or soluble FasL will generally be of human origin although genes from other closely related species that exhibit high homology and biologically identical or equivalent function in humans may be used, if the gene product does not induce an adverse immune reaction in the recipient.
  • a reporter or marker gene can be included in the gene delivery vehicle to facilitate identification of those cells into which the vehicle is successfully inco ⁇ orated (Kass-Eisler, et al. (1994) Gene Therapy
  • marker genes may prove especially helpful. Screening markers or reporter genes are genes that encode a product that can readily be assayed. Non-limiting examples of screening markers include genes encoding for green fluorescent protein (GFP) or genes encoding for a modified fluorescent protein. Preferably, the marker gene included in the delivery vehicle is a selectable marker.
  • GFP green fluorescent protein
  • the marker gene included in the delivery vehicle is a selectable marker.
  • a "positive" selectable marker gene encodes a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neo r ) gene are resistant to the compound G418. Cells that do not carry the Neo r gene marker are killed by G418. Negative selectable marker genes encode a product that allows cells expressing that product to be selectively killed. For example, as described above the conditionally activated cytotoxic agent may also be a selectable marker such as HSV-tk. Cells expressing this gene can be selectively killed using gancyclovir or acyclovir.
  • FasL proteins and polypeptides of this invention are obtainable by a number of processes well known to those of skill in the art, which include purification, chemical synthesis and recombinant methods.
  • Full length FasL protein can be purified from a FasL + cell or tissue lysate using the process described below or by methods such as immunoprecipitation with anti-FasL antibody, and standard techniques such as gel filtration, ion-exchange, reversed-phase, and affinity chromatography using a FasL fusion protein as shown herein.
  • standard techniques such as gel filtration, ion-exchange, reversed-phase, and affinity chromatography using a FasL fusion protein as shown herein.
  • this invention also provides the processes for obtaining the proteins and polypeptides useful this invention as well as the products obtainable and obtained by these processes.
  • compositions containing the polypeptides and proteins for use in this invention including compositions comprising a pharmaceutically acceptable carrier, are further provided by this invention.
  • proteins and polypeptides also can be obtained by chemical synthesis using a commercially available automated peptide synthesizer such as those manufactured by
  • this invention also provides a process for chemically synthesizing the proteins of this invention by providing the sequence of the protein and reagents, such as amino acids and enzymes and linking together the amino acids in the proper orientation and linear sequence.
  • the proteins and polypeptides can be obtained by well-known recombinant methods as described, for example, in Sambrook et al. (1989) Supra, using the host cell and vector systems described and exemplified below.
  • This invention further provides a process for producing a FasL protein, analog, mutein or fragment thereof, by growing a host cell containing a polynucleotide encoding the mammalian protein, the polynucleotide being operatively linked to a promoter of RNA transcription.
  • the host cell is grown under suitable conditions such that the polynucleotide is transcribed and translated into protein and purifying the protein so produced.
  • the potentially therapeutic agent identified by this method is an agent that is an agonist or antagonist of the localized FasL induced proinflammatory response, tumor regression, anti-infective therapy or localized neutrophil infilitration.
  • an "agent” is intended to include, but not be limited to, a small organic molecule, a compound, a composition, a DNA molecule, an RNA molecule, a protein, a polypeptide, an antibody, an antibody fragment, an anti-idotypic antibody or fragment or a fusion protein. It should be understood, although not always explicitly stated that the agent is used alone or in combination with another agent, having the same or different biological activity as the agents identified by the inventive screen. The agents and methods are also intended to be combined with other therapies.
  • suitable cell cultures or tissue cultures are first provided.
  • the cell is a cultured cell or alternatively, the cells can be from a tissue biopsy.
  • the cells are cultured under conditions (temperature, growth or culture medium and gas (CO 2 )) and for an appropriate amount of time to attain exponential proliferation without density dependent constraints. More than one test and control culture is provided, one containing neutrophils and the other free of neutrophils.
  • suitable cells may be cultured in microtiter plates and several agents may be assayed at the same time by noting phenotypic changes or cell death.
  • a suitable cell as defined above is contacted with the agent to be tested either prior to, concurrently, or after the FasL polynucleotide or protein has been contacted with a sample containing the suitable cells.
  • a positive and negative control sample should be run simultaneously with the test sample.
  • the positive control will only receive the FasL polynucleotide or protein.
  • the negative control will not receive FasL polynucleotide or protein or the test agent.
  • the assay can be run in the presence and absence of neutrophils to determine if the effect, if any, is the result of the presence of neutrophils and thus, the inflammatory response.
  • the use of the negative control as compared to the test sample and the positive control allows one of skill in the art to compare the activity of the test agent versus the activity of the agent as another agent having FasL gene product activity.
  • the identification of this activity allows one to find alternative therapies to the FasL polynucleotide or protein.
  • the agent is contacted with the cells under conditions which inhibit the proliferation or death of the cells and/or the induction of a proinflammatory response.
  • the agent is a composition other than a DNA or RNA nucleic acid molecule
  • the suitable conditions may be directly adding the agent to the cell culture or added to culture medium.
  • an "effective" amount must be added which can be empirically determined.
  • the protein or polypeptide is administered to the cells, in vitro or in vivo, as described above.
  • the test cell is grown in small multi-well plates and is used to detect the biologic activity of test drugs.
  • the successful candidate drug will block (antagonist) or promote (agonist) the slowing of growth or kill the test cell type. It also may block (antagonist) or promote (agonist) the induction of the proinflammatory response provoked by FasL.
  • Kits containing the agents and instructions necessary to perform the screen in vitro and the methods as described herein are further provided by this invention.
  • the agents identified herein as effective for their intended pu ⁇ ose can be administered to subjects or individuals susceptible to or at risk of developing a disease correlated to the presence of unwanted proliferation of normal or malignant cells, such as cancer.
  • the agent When the agent is administered to a subject such as a mouse, a rat or a human patient, the agent can be added to a pharmaceutically acceptable carrier and systemically or topically administered to the subject.
  • a tumor sample is removed from the patient.
  • Therapeutic amounts can be empirically determined and will vary with the pathology being treated, the subject being treated and the efficacy and toxicity of the agent. When delivered to an animal, the method is useful to further confirm efficacy of the agent.
  • mice groups of nude mice (Balb/c NCR nu/nu female, Simonsen, Gilroy, CA) are each subcutaneously inoculated with about 10 5 to about 10 9 hype ⁇ roliferative, cancer or target cells as defined herein.
  • the agent is administered, for example, by subcutaneous injection around the tumor. Tumor measurements to determine reduction of tumor size are made in two dimensions using venier calipers twice a week. Other animal models may also be employed as appropriate.
  • Renca renal epithelial carcinoma cell line was studied.
  • Another cell line, the FasL " CT26 colon carcinoma was studied in comparison. Renca cell lines, in contrast to the CT26, were susceptible to lysis by FasL in a chromium release assay.
  • an adenoviral vector encoding mouse FasL, ADV-FasL was used. Because the producer line, 293 cells, expresses FasL and is susceptible to FasL, a FasL-resistant clone of 293 cells was selected and used to produce this vector.
  • ADV-FasL was also prepared in normal 293 cells by inclusion of a known peptide inhibitor of ICE- like proteases, zVAD-fmk.
  • Infection of Renca cells by ADV-FasL caused massive cell death in vitro ( Figure 1).
  • Two days after infection, >95%> of Renca cells were nonviable and the majority appeared positive in the deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay, which detects DNA strand breaks and is diagnostic for cells undergoing apoptosis, in contrast to ADV- ⁇ EI vector controls.
  • TUNEL deoxynucleotidyl transferase-mediated dUTP nick end labeling
  • FasL was confirmed by FACS analysis in these cells after ADV-FasL infection using FasL-Fc fusion protein, suggesting that expression of FasL and its engagement of FasL induced this apoptotic cell death.
  • Renca cells were inoculated subcutaneously into the flanks of syngeneic, Balb/C mice. After nodules were established (-0.5 cm), tumors were injected with phosphate buffered saline (PBS), ADV-
  • CT26-FasL cells failed to form tumors in syngeneic recipients, in contrast to CT 26-neo control cells ( Figure 3B).
  • these lines were inoculated into immunodeficient mouse strains, including nude (Giovanella, B. C. and Fogh, J. (1985) Adv. Cancer Res. 44:69), severe combined immunodeficient (SCID) (Bosma, G. C. et al. (1983) Nature 301:527) or SCID-beige (Roder, J. and Duwe, A. (1979) Nature 278:451) mice which are deficient in T, B, and ⁇ K cell activities.
  • nude Gaovanella, B. C. and Fogh, J. (1985) Adv. Cancer Res. 44:69
  • SCID severe combined immunodeficient mice
  • SCID-beige mice which are deficient in T, B, and ⁇ K cell activities.
  • FasL expression in melanoma tumor nodules was found in connective tissue and not associated with tumor cell membranes. Hahne, M. et al. (1996) Science 274:1363.
  • FasL expression reported previously in malignancies is unlikely to be derived from expression on transformed cells, but rather appears to be derived from different cell types and is matrix-associated in vivo where it may exert different, presumably immunosuppressive, effects.
  • Renca and CT26 were obtained from the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, U.S.A., and grown in RPMI 1640 medium supplemented with 10%> fetal calf serum (FCS), L-glutamine, and antibiotics.
  • HepG2 a human hepatocellular carcinoma cell line, and Hela cell, a human cervical carcinoma cell line, were obtained from the ATCC.
  • Human glioma cell lines (G87, G138, G373) were a generous gift from Dr. P. Kish and Dr. K. Murazko.
  • Human melanoma cell lines (M316, M342, M347, M444, M449, M720) were a generous gift from Dr. A. Chang.
  • the melanoma cells were grown in RPMI 1640 supplemented with 10% >
  • FCS FCS, L-glutamine, and antibiotics. Others were grown in DMEM with 10%) FCS, L-glutamine, and antibiotics.
  • FasL was synthesized from mouse spleen poly(A) RNA by reverse transcription (RT)-PCR using Superscript II reverse transcriptase (Gibco BRL, Gaithersburg, MD) and ExpandTM long template PCR system (Boehringer Mannheim, Germany) (sense primer: AATTGTCGACCACCATGGAGCAGCCCATGAATTACCATGTCCC, antisense primer: AATTGGATCCTCTAGATTAAAGCTTATACAAGCCGAAAAAGGT
  • the FasL cDNA was inserted into the mammalian expression vector pVR1012 neo (Vical, Inc., San Diego, CA) harboring CMV enhancer/promoter, bovine growth hormone poly(A) signal (Plautz, G.E. et al. (1993) Proc. Natl. Acad. Sci. USA 90:4645) and neomycin resistant gene as a selection marker.
  • CT26 cells were cultured in RPMI 1640 media (Gibco BRL) with 10% fetal calf serum (FCS).
  • CT26 cells were transfected with linearized FasL/pVR1012 neo plasmid by electroporation using Gene pulser (Bio-rad, Hercules, CA), and transfected cells were selected with 1 mg/ml of Geneticin (Gibco BRL).
  • Gene pulser Bio-rad, Hercules, CA
  • Gibco BRL Geneticin
  • CT26-FasL The CT26 cell line which expresses FasL stably (CT26-FasL) was generated as reported in Nagata, S. and Goldstein, P. (1995) Science 267:1449.
  • the complete amino acid and nucleic acid sequence coding for FasL is provided in Figure 6 and SEQ ID NO: 1 and 2.
  • the mouse FasL cDNA was inserted into a mammalian expression vector which utilizes the CMV enhancer/promoter, bovine growth hormone poly-A signal, and neomycin resistant gene as a selection marker.
  • CT26 cells were transfected with this plasmid by electroporation and were selected with 1 mg/ml of Geneticin (GIBCO BRL, Gaithersburg, MD).
  • GEBCO BRL Gaithersburg, MD
  • a clone which expressed FasL at high levels was isolated by limiting dilution.
  • CT26-neo was prepared in the same way.
  • Target cells (1 x 10 6 ) were stained with anti-Fas antibody (Pharmingen, San Diego, CA) or isotype control IgG followed by FITC-conjugated anti-IgG second antibody (Pharmingen), or Fas-Fc fusion protein (described below) followed by FITC-conjugated antibody to the Fc fragment of IgG (Jackson Immunoresearch Lab, West Grove, PA) to detect the expression of Fas or FasL, respectively.
  • conditioned medium from vector-control transfected 293 cells was used as a negative control. Relative fluorescence intensity was measured by FACS analysis.
  • CT26 cells transfected with pVR1012 neo backbone (CT26-neo) were prepared for the control at the same time.
  • Fas-Fc fusion protein cDNAs encoding the mouse FAS extracellular domain and the human IgG Fc portion as described in Ellison, J.W. et al. (1982) Nucl. Acids. Res. 10:4071, were synthesized by RT-PCR FAS sense primer: AATTGTCGACCACCATGGTGTGGATCTG GGCTGTCCTGCCTCTG, antisense primer: AATTGGATCCTCGAGGCGA
  • chimeric cDNA was inserted into pVR1012 neo backbone and transfected into 293 cells by the calcium-phosphate method. On day 7 post-transfection, the conditioned medium was collected, filtered and used in flow cytometric [fluorescense-activated cell sorting (FACS)] analysis.
  • FACS fluorescense-activated cell sorting
  • FasL-induced cytotoxicity assays FasL-mediated cytotoxicity was assayed essentially as described below.
  • Jurkat cells (1 x 10 6 ) were incubated for 2 hours at 37°C with 20 ⁇ Ci of [ 51 Cr] sodium chromate (Amersham, Little Chalfont, Buddinghamshire, UK) in 100 ⁇ l of RPMI 1640 containing 10%) FCS. After washing with medium, the cells were used as the target.
  • the [ 5, Cr]- labeled target cells (1 x 10 4 ) were mixed with CT26-FasL or CT26-neo (the indicated effector target rations) in round-bottomed microtiter plates in a total volume of 200 ⁇ l.
  • the plates were centrifuged at 700 ⁇ m for 2 minutes and incubated for 4 hours at 37°C in 5%> CO 2 . After the centrifugation at 1200 rmp for 5 minutes, the supernatants were collected with the harvesting frame (Skatron Instruments, Inc., Sterling, VA) and assayed for radioactivity.
  • the spontaneous release of [ 51 Cr] was determined by incubating the target cells with the medium alone, whereas the maximum release was determined by incubating in 0.1 %> Triton X-100.
  • the specific lysis (%>) was calculated as: (experimental [ 51 Cr] release - spontaneous [ 51 Cr] release)/(maximum [ 51 Cr] release - spontaneous [ 5I Cr] release). The ratio of spontaneous 5, Cr release to maximum [ 5l Cr] release was between 11.1% and 17.6%.
  • the recombinant adenoviral vector, ADV-FasL was prepared by homologous recombination between sub360 genomic DNA, an Ad5 derivative with a deletion in the E3 region, and a FasL expression plasmid, pAd-FasL, described above.
  • the pAd-FasL encodes the mouse FasL cDNA under control of the cytomegaloviral enhancer/promoter and has a deletion in the El A and E1B region, impairing the ability of this virus to replicate and transform nonpermissive cells.
  • the presence of FasL cDNA and absence of the El in this viral genome was confirmed by Southern blot analysis.
  • ADV-FasL The construction and propagation of ADV-FasL were performed in the FasL-resistant clone of 293 cells which was isolated by successive FasL transfections followed by limiting dilution.
  • This 293 clone exhibited low expression of FAS by FACS analysis and relative resistance to FasL- stimulation in [ 5 l Cr] assays or by inclusion of a Caspase inhibitor, N-benzyloxycarbonyl Val-Ala-Asp-flouromethylketone (2-VAD-fmk) in the cell culture media.
  • the 293 cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10%> FCS, L-glutamine and antibiotics.
  • DMEM Dulbecco's modified Eagle's medium
  • Cesium chloride-purified virus was dialysed against PBS and diluted for storage in a 13%> glycerol-PBS solution to yield a final concentration of lxlO 12 viral particles/ml. All stocks were sterilized by passage through a 0.45 ⁇ m filter and evaluated for the presence of replication-competent virus. In the plaque assay, 0.9xl0 2 particles corresponded to 1 plaque forming unit on 293 cells.
  • mice Six-week-old female Balb/C, SCID, and SCID-beige (CB17- origin) mice were obtained from Charles River (Wilmington, MA) and Taconic (Germantown, NY), respectively, and kept in specific-pathogen-free environment. Cells were harvested with trypsin, incubated in growth medium at 37°C for one hour to recover surface molecules, washed three times with PBS, and resuspended in PBS. The CT26-neo and CT26-FasL cells (5 x 10 ⁇ ) were inoculated subcutaneously in the flank.
  • Tumor size was followed in two pe ⁇ endicular dimensions using calipers.
  • 50 ⁇ l of viral solution (lxlO 12 particle/ml) was injected into the tumor with a 26-gauge hypodermic needle after the tumor mass was established (-0.5 cm).
  • the histology of the major organs was examined by microscopic observation of hematoxylin and eosin stained slides by an experienced pathologist (D.G.).
  • Fresh-frozen tissues of CT26-FasL and CT26-neo tumor on day 2 were fixed with acetone.
  • the section was first incubated with anti-Ly-6G (GR-1) monoclonal antibody (RB6-8C5) (Pharmingen) or an istotype control rat IgG.
  • Biotinylated anti-rat IgG2b second antibody (Pharmingen) was added followed by the addition of preformed avidin biotinylated horseradish peroxidase complex.
  • the signal was visualized by incubation in peroxidase substrate.
  • the RB6-8C5 antibody reacts mainly with neutrophils and, to a lesser extent, with activated monocytes/macrophage (see Garrone, P. et al. (1995) J. Exp.
  • HepG2 cells HepG2
  • Hela cells Hela
  • three glioma cell lines G87,G138,G373
  • M316, M342, M347, M444, M449, M720 six melanoma cell lines established from human malignancies were labeled with [ 51 Cr] and co-incubated with CT26-neo (open bar) or CT26-FasL (solid bar) at an effector/target ratio of 5 for
  • FasL + renal carcinoma 4 hours, as described previously in Arai, H. et al. (1997) Nat. Med., Supra, and represent mean and standard deviation in three assays.
  • Expression of FasL in each cell line was examined by FACS analysis using anti-human FasL antibody (Pharmingen) or isotype- control mouse IgG followed by FITC-conjugated anti-mouse IgG second antibody (Pharmingen).
  • FITC-conjugated anti-mouse IgG second antibody Pharmingen
  • Renca is presented (right).
  • Figure 5B shows expression of FasL and cytotoxicity to Jurkat cells in human malignancies.
  • a lysis assay was performed using [ 51 Cr]-labeled Jurkat target cells, which are susceptible to FasL.
  • the indicated tumor cells were cocultivated at an effector/target ratio of 5 for 4 hours.
  • FACS analysis was performed using a FasL-Fc fusion protein and

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Abstract

L'invention concerne un procédé servant à inhiber la prolifération d'une cellule ou d'un pathogène par administration au locus de la cellule d'une quantité efficace de FasL. Elle concerne également un procédé servant à identifier des agents modulant la stimulation par FasL d'une réaction proinflammatoire localisée.
PCT/US1998/014770 1997-07-17 1998-07-16 Procedes et compositions servant a reduire une tumeur WO1999003998A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2002018593A3 (fr) * 2000-08-29 2002-12-12 Bioniche Life Sciences Inc Modulation de l'expression de fas et de fasl
US20140294754A1 (en) * 2011-04-14 2014-10-02 Jaime Modiano Use of tumor fas expression to determine response to anti-cancer therapy
WO2023034796A1 (fr) * 2021-08-31 2023-03-09 Yale University Régulateurs de tension cytosquelettique et compositions de combinaison de ligands fas et méthodes de traitement les utilisant

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* Cited by examiner, † Cited by third party
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US8991401B2 (en) 2008-03-31 2015-03-31 Lenticular Research Group, Llc Processes and apparatus for preventing, delaying or ameliorating one or more symptoms of presbyopia

Family Cites Families (2)

* Cited by examiner, † Cited by third party
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AU4056089A (en) * 1988-07-20 1990-02-19 Amgen, Inc. Method of treating inflammatory disorders by reducing phagocyte activation
EP0620739A4 (en) * 1992-09-15 1997-01-15 Immunex Corp Method of treating tnf-dependent inflammation using tumor necrosis factor antagonists.

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ARAI H ET AL: "Gene transfer of Fas ligand induces tumor regression in vivo.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, (1997 DEC 9) 94 (25) 13862-7. JOURNAL CODE: PV3. ISSN: 0027-8424., United States, XP002084131 *
GRIFFITH T S ET AL: "Fas ligand -induced apoptosis as a mechanism of immune privilege [see comments].", SCIENCE, (1995 NOV 17) 270 (5239) 1189-92. JOURNAL CODE: UJ7. ISSN: 0036-8075., United States, XP002084130 *
KANG S-M ET AL: "Rapid neutrophilic destruction of Fas ligand expressing cell lines in vivo reveals a novel proinflammatory function of Fas ligand.", SURGICAL FORUM 48 (0). 1997. 451-453. ISSN: 0071-8041, XP002084132 *
SEINO K ET AL: "Antitumor effect of locally produced CD95 ligand.", NATURE MEDICINE, (1997 FEB) 3 (2) 165-70. JOURNAL CODE: CG5. ISSN: 1078-8956., United States, XP002084129 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002018593A3 (fr) * 2000-08-29 2002-12-12 Bioniche Life Sciences Inc Modulation de l'expression de fas et de fasl
US20140294754A1 (en) * 2011-04-14 2014-10-02 Jaime Modiano Use of tumor fas expression to determine response to anti-cancer therapy
US10352936B2 (en) * 2011-04-14 2019-07-16 Apoplogic Pharmaceuticals, Inc. Use of tumor Fas expression to determine response to anti-cancer therapy
WO2023034796A1 (fr) * 2021-08-31 2023-03-09 Yale University Régulateurs de tension cytosquelettique et compositions de combinaison de ligands fas et méthodes de traitement les utilisant

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