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WO1997034501A1 - Metalloproteases dirigees sur un recepteur de folate et leurs procedes d'utilisation - Google Patents

Metalloproteases dirigees sur un recepteur de folate et leurs procedes d'utilisation Download PDF

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Publication number
WO1997034501A1
WO1997034501A1 PCT/US1997/004595 US9704595W WO9734501A1 WO 1997034501 A1 WO1997034501 A1 WO 1997034501A1 US 9704595 W US9704595 W US 9704595W WO 9734501 A1 WO9734501 A1 WO 9734501A1
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cells
metalloenzyme
cell
folate receptor
metalloprotease
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PCT/US1997/004595
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English (en)
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Asok Antony
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Advanced Research & Technology Institute
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Priority to AU25398/97A priority Critical patent/AU2539897A/en
Publication of WO1997034501A1 publication Critical patent/WO1997034501A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates generally to the fields of enzymology, biochemistry and cell biology More specifically, the invention relates to metalloproteases, and in particular to metalloproteases having activity for cleaving hydrophobic folate receptors to hydrophilic forms, and to assays which can be used to detect and quantify the presence of such metalloproteases
  • Folates which are important precursors of coenzymes in reactions necessary for DNA synthesis are essential for proliferating cells
  • the shut-down of DNA synthesis and one-carbon metabolism arising from folate deficiency perturbs the cell cycle resulting in adverse clinical outcomes from megaloblastosis (Antony, 1995)
  • induction of a functional folate deficiency with antifolates is desirable in certain target cells (e.g., in leukemia, cancers, and against immunocompetent cells) Acquisition of folate is therefore critical to the viability of normal and malignant cells
  • the reduced-folate carrier which is related to glucose transporters (Dixon el al., 1994) is a low affinity, high capacity system that mediates uptake of reduced-folates into cancer cells predominantly at pharmacological (micromolar, ⁇ M) extracellular folate concentrations
  • Cellular folate transport also can be mediated by
  • FR membrane-associated folate receptors
  • FR- ⁇ Three human FR cDNA isoforms, FR- ⁇ , - ⁇ , and - ⁇ (Antony, 1996) have been cloned
  • Most studies related to transfection/transduction of FR cDN A into cells have used FR- ⁇ into cervical carcinoma (HeLa-IUi) cells gives rise to a glycosyl-phosphatidylinositol (GPI)-anchored protein similar to native FR (Sun et al, 1995, Verma et al, 1992, Luhrs and Slomiany, 1989)
  • GPI glycosyl-phosphatidylinositol
  • the corresponding FR are found on the cell surface as glycoproteins which bind physiologic serum 5- methyltetrahydrofolate with high affinity and transport the vitamin into cells (Antony, 1992)
  • PFR Human placental FR
  • GPI glycosyl-phosphatidylinositol
  • hydrophilic PFR released into the growth media of chorionic villi identified a species which was much smaller, based on amino acid analysis (22.5 kD), when compared to native PFR, but was of comparable M r to the soluble folate binding protein isolated from human milk (Antony et al, 1982) and the growth media of KB cells (Elwood et al, 1986).
  • FR in KB cells are GPI-anchored (Luhrs and Slomiany, 1989) in the membranes of another KB cell line.
  • Biosynthetically-labeled [ 3 H]leucine-hydrophobic FR was found to contain a full-length FR polypeptide that is leucine-rich in its C- terminus (Elwood et al, 1991) These investigators identified an activity in washed,
  • the unique KB cell FR differed considerably from PFR which, like other GPI-anchored proteins, which has lost its C-terminal hydrophobic polypeptide during post ⁇ translational addition of the performed GPI anchor (Antony and Miller, 1994, Udenfriend and Kodukula, 1995)
  • the putative placental metalloprotease which endoproteolytically cleaved the FR polypeptide substrate from KB cells (Elwood et al, 1991), is the same enzyme which converts mature GPI-linked PFR (Verma et al, 1992, Verma and Antony, 1991) to soluble forms
  • compositions and methods relating to FR-directed metalloproteases In particular, protein compositions, purified metalloproteases, nucleic acids encoding metalloproteases, antibodies specific for metalloproteases and methods of using the preceding both in vitro and in vivo are contemplated
  • the present invention provides a method for purifying a folate receptor-directed metalloprotease (FR-MP) comprising the steps of providing a source of metalloenzyme-rich material, mixing the metalloenzyme-rich material with a detergent, isolating a micellar phase containing metalloenzyme; and purifying FR-MP from the micellar phase
  • the metalloenzyme-rich material is human placental tissue
  • the human placental is homogenized, centrifuged to yield a supernatant which is separated therefrom
  • the detergent may be a non-ionic detergent
  • the non-ionic detergent is Triton X-l 14TM
  • the metalloenzyme-detergent mixture is subjected to temperature-induced phase separation at 30°C
  • the isolating step may comprise affinity chromatography and concentration
  • the purifying comprises high performance liquid chromatography to purify the FR-MP to homogeneity
  • Another embodiment of the present invention describes a method of purifying folate receptor-directed metalloprotease (FR-MP) comprising the steps of providing a metalloenzyme-rich material, mixing the metalloenzyme-rich material with a detergent, temperature-inducing phase separation of the metalloenzyme-detergent mixture, subjecting the micellar fraction of the metalloenzyme-detergent mixture to affinity chromatography, whereby an affinity chromatography eluate is formed, subjecting the affinity chromatography eluate to concentration by ultrafiltration to whereby a concentrate is formed, and subjecting the concentrate to reverse-phase high- performance liquid chromatography, whereby a homogenous preparation of FR-MP is formed
  • FR-MP folate receptor-directed metalloprotease
  • the present invention further discloses a folate receptor-directed metalloprotease purified according to a process having the steps of providing a metalloenzyme-rich material, mixing the metalloenzyme-rich material with a detergent, temperature-inducing phase separation of the metalloenzyme-detergent mixture, subjecting the micellar fraction of the metalloenzyme-detergent mixture to affinity chromatography, whereby an affinity chromatography eluate is formed, subjecting the affinity chromatography eluate to concentration by ultrafiltration to whereby a concentrate is formed, and subjecting the concentrate to reverse-phase high- performance liquid chromatography
  • the present invention also further provides a folate receptor-directed metalloprotease (FR-MP) preparation prepared according to a process having the steps of providing a metalloenzyme-rich material, mixing the metalloenzyme-rich material with a detergent; temperature-inducing phase separation of the metalloenzyme- detergent mixture; subjecting the micellar fraction of the metalloenzyme-detergent mixture to affinity chromatography, whereby an affinity chromatography eluate is formed, subjecting the affinity chromatography eluate to concentration by ultrafiltration to whereby a concentrate is formed; and subjecting the concentrate to reverse-phase high-performance liquid chromatography.
  • FR-MP folate receptor-directed metalloprotease
  • FR-MP folate receptor-directed metalloprotease
  • FR-MP folate receptor-directed metalloprotease
  • FR-MP folate receptor-directed metalloprotease
  • a nucleic acid encoding a folate receptor-directed metalloprotease is also contemplated.
  • the nucleic acid may be in an expression vector comprising a DNA encoding a folate receptor-directed metalloprotease operably linked to a promoter wherein the DNA is in an antisense orientation to the promoter.
  • the present invention provides a recombinant host cell comprising a DNA encoding a folate receptor-directed metalloprotease operably linked to a promoter.
  • the present invention discloses a method of detecting folate receptor-directed metalloprotease (FR-MP) activity in a sample comprising the steps of providing a hydrophobic placental folate receptor (PFR); contacting the PFR with the sample; and determining the presence or absence of hydrophilic folate receptor.
  • the determining comprises detergent-aqueous phase separation of PFR.
  • the detergent used is Triton X-l 14TM.
  • the PFR may be labeled or unlabeled. In those aspects where the PFR is labeled, the label is selected from the group consisting of a radiolabel, a fluorescent label, a chemiluminescent label and an enzyme
  • the present invention still further provides a method for detecting folate receptor-directed metalloprotease (FR-MP) comprising the steps of providing a sample, contacting the sample with an antibody that binds immunologically to FR-MP, and determining the binding of the antibody to the sample
  • the antibody may be labeled
  • the label may be selected from the group consisting of a radiolabel, a fluorescent label, a chemiluminescent label and an enzyme
  • the sample may be purified prior to the contacting
  • the purification comprises at least one of centrifugation, detergent phase separation, affinity chromatography, ultrafiltration, high-performance liquid chromatography and electrophoresis
  • Also contemplated is a method for removing surface-bound folate receptor from a cell comprising the steps of providing a purified folate receptor-directed metalloprotease (FR-MP), and contacting the FR-MP with the cell, whereby the FR- MP cleaves surface-bound folate receptor
  • FR-MP purified folate receptor-directed metalloprotease
  • Another aspect of the invention is to provide a method for reducing surface- bound folate on a cell comprising the steps of providing an expression construct comprising a DNA encoding a folate receptor-directed metalloprotease (FR-MP) operably linked, in a sense orientation, to a promoter, and contacting the expression construct with the cell under conditions facilitating the uptake of the expression construct by the cell
  • the expression construct is a viral vector selected from the group consisting of retrovirus, adenovirus, adeno-associated virus, herpesvirus and vaccinia virus
  • the present invention further provides a method for increasing surface-bound folate on a cell comprising the steps of providing a folate receptor-directed metalloprotease (FR-MP) DNA operably linked, in an antisense orientation, to a promoter, and contacting the DNA with the cell under conditions facilitating the uptake of the DNA by the cell.
  • FR-MP folate receptor-directed metalloprotease
  • the DNA may contain a coding region or a non-coding region or both.
  • the DNA comprises intron-exon junction.
  • the DNA comprises a transcription start site.
  • the DNA also may comprise a translation start site or be part of an expression construct.
  • the present invention discloses a method of protecting a cell in a patient from a folate receptor-targeted therapy comprising the steps of providing an expression construct comprising a DNA encoding a folate receptor- directed metalloprotease (FR-MP) operably linked, in a sense orientation, to a promoter; contacting the expression construct with the cell under conditions facilitating the uptake of the expression construct by the cell; and administering a folate-receptor targeted therapeutic to the patient.
  • the expression construct may be a viral vector selected from the group consisting of retrovirus, adenovirus, adeno- associated virus, herpesvirus and vaccinia virus
  • a method of protecting a cell in a patient from folate receptor-targeted therapy also is contemplated .
  • the method comprises the steps of providing an antibody that binds immunologically to membrane bound folate a receptor; and administering a folate receptor targeted therapeutic to the patient.
  • the present invention provides a method of protecting a cell in a patient from a folate receptor-targeted therapy comprising the steps of providing an expression construct comprising a DNA encoding a folate receptor operably linked, in an antisense orientation, to a promoter; contacting the expression construct with the cell under conditions facilitating the uptake of the expression construct by the cell; and administering a folate-receptor targeted therapeutic to the patient.
  • FIGS. IA and IB Rate of conversion of hydrophobic PFR to hydrophilic PFR by crude metalloenzyme as a function of (FIG IA) dose and (FIG IB) incubation time.
  • FIG IA Two tubes containing 9 04 pmol of [ 3 H]PteGlu- labeled hydrophobic PFR, increasing concentrations of crude solubilized metalloenzyme was added in the absence (closed circles) and presence (open circles) of 60 mM EDTA After incubation at 37°C and temperature-induced phase-separation at the cloud point of Triton X-l 14, the aqueous phase containing hydrophilic PFR (product) was separated from the substrate retained in the micellar phase, and the percent conversion was determined.
  • FIG IB Two concentrations of detergent-rich metalloenzyme, 0 4 mg (closed squares) and 2 mg (open squares), were incubated with 9 04 pmol of [ 3 H]PteGlu- labeled
  • FIGS. 2A-C Reverse-phase HPLC. SDS-PAGE. and functional analysis of the purified metalloproteases
  • FIG. 2A The HPLC-purified metalloenzyme (eluted by 51% buffer B) was re-analyzed under similar conditions by reverse- phase HpLC and each fraction was spectrophotometrically analyzed for protein
  • FIG 2B SDS-PAGE (7 5%) of reverse-phase HPLC-purified metalloenzyme before (lane J) and after (lane 2) deglycosylation with recombinant glycopeptidase F Each well was loaded with 15 ⁇ g of protein and stained with Coomassie Blue
  • FIG. 2C Dose-response curve of the purified sample using the temperature-induced phase-separation assay in Triton X-l 14
  • FIGS. 3A-F Analysis of various parameters of metalloprotease activity
  • FIG. 3A Rate of conversion of 125 I-hydrophobic PFR to hydrophilic PFR as a function of time
  • Metalloenzyme 60 ⁇ g in 100 ⁇ l of 10 mM potassium phosphate, pH 7 5, containing 20 mM MgCl 2 was incubated with 125 I- hydrophobic PFR (350 fmol) for various times indicated in the absence or presence of 60 mM EDTA
  • Each data point represents the mean of studies carried out in duplicate; there was ⁇ 5% variation from the mean in more than 3 comparable studies carried out with different preparations
  • FIG 3B Dose- response curves using a fixed concentration of 125 I-hydrophobic PFR (350 fmol) and increasing concentrations of purified metalloenzyme in the absence and presence of EDTA
  • FIG. 3A Rate of conversion of 125 I-hydrophobic PFR to hydrophilic PFR as a function of time
  • FIGS. 3D-F Characteristics of inhibition with 1,10-phenanthroline (FIG 3D), and reactivation of metalloenzyme (50 ⁇ g) with increasing concentrations of various cations ((MgCl 2 , MnCl 2 , CaCl , ZnCl 2 ) after inhibition with 60 mM EDTA (FIG. 3E) and 60 mM EGTA (FIG. 3F).
  • FIG. 4 Fluorescence activated cell sorting analysis of cultured human cervical carcinoma cells Cells were reacted with nonimmune serum (solid lines), or
  • FIG. 5 Relationship between FR expression (gene dose) and TK activity in single cell-derived clones from sense and antisense cells The cells transduced with sense and antisense FR cDNA from Sun et al. (1995) were analyzed for
  • TK activity Quadruplicate samples of 1 x IO 8 cells from each cell line were cultured in 500 cm 2 800 ml capacity three-tier Nunclon flasks (A/S Nunc, Roskilde, Denmark) and harvested during logarithmic growth phase at -70- 80% confluency. TK (EC 2.7.1.21) was assayed as described (Weber et al, 1977). TK activity was defined as the amount of the enzyme required to convert one nmol of thymidine to dTMP per h.
  • FR-MP folate receptor-metalloprotease
  • An FR-MP ofthe present invention in one embodiment, has a molecular weight of about 63,000 daltons before deglycosylation and 55,000 to 60,000 daltons after deglycosylation. In a preferred embodiment it has a molecular weight of 58,000 daltons after deglycosylation.
  • Metalloproteases of the invention are activated by divalent cations, for example the metalloprotease studied below is activated by Mg 2+ , Ca 2+ , Mn 2+ and Zn 2+ , unlike the KB cell FR-directed metalloprotease (Elwood et al, 1991), which is not activated by Mg 2+ .
  • the purification involved a series of steps which included the steps of (1) preparing a metalloenzyme-rich supernatant; (2) temperature-inducing phase separation of the supernatant in the presence of a detergent to form a detergent-rich micellar phase enriched in the metalloenzyme; (3) subjecting the contents of the micellar phase to affinity chromatography to prepare an eluate containing the metalloenzyme; (4) concentrating the eluates from step (3) by ultrafiltration to form a metalloprotease concentrate, and (5) subjecting the metalloprotease concentrate to reverse-phase HPLC so as to recover a fraction comprised of a substantially homogeneous preparation of the metalloprotease.
  • the metalloprotease was hydrophobic, including the fact that the crude EDTA-sensitive metalloprotease was solubilized from placental membranes with Triton XI 14TM and then recovered in the micellar phase phase at the cloud point of that detergent.
  • the metalloprotease was eluted by hydrophobic elution buffers from reverse-phase HPLC, and both the HPLC-purified non-iodinated and iodinated metalloprotease sequestered in the micellar.
  • Further evidence for the membrane localization was the presence of cross-reacting moieties in close proximity to its hydrophobic substrate on normal and malignant cells.
  • the reverse-phase HPLC-isolated FR-MP which exhibited biological activity in converting hydrophobic PFR to hydrophilic forms met several criteria for purity. First, it exhibited a single protein peak on reverse-phase HPLC, and a single band of protein staining on SDS-PAGE. Second, when this preparation was iodinated and similarly analyzed, there was only a single iodinated species. Interestingly, immunofluorescence indicated that moieties sharing epitopes with placental metalloprotease were localized on plasma membranes of normal and malignant cells in a similar distribution as the hydrophobic FR substrate.
  • compositions include genes encoding FR-MP and antibodies specific for an FR-MP.
  • metalloproteases having activity against folate receptor there has been identified metalloproteases having activity against folate receptor
  • the term metalloprotease is well-known to those of skill in the art Because of their activity against FR, the metalloproteases of the present invention are designated FR-MP
  • the present invention also relates to fragments of FR-MP 's that may or may not retain the protease (or other) activity. Fragments including the N- and C-termini of the molecule may be generated by genetic engineering of translation stop sites within the coding region (discussed below) Alternatively, treatment of a FR-MP with other proteases can produces a variety of N- terminal, C-terminal and internal fragments Examples of fragments may include 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 75, 80, 85, 90, 95, 100, 200, 300, 400 or more amino acids in length
  • fragments may be purified according to known methods, such as precipitation (e.g., ammonium sulfate), HPLC, ion exchange chromatography, affinity chromatography (including immunoaffinity chromatography) or various size separations (sedimentation, gel electrophoresis, gel filtration)
  • variants of FR-MP Amino acid sequence variants of the polypeptide can be substitutional, insertional or deletion variants
  • Deletion variants lack one or more residues of the native protein which are not essential for protease function or immunogenic activity, and are exemplified by the variants lacking a transmembrane sequence described above
  • Another common type of deletion variant is one lacking secretory signal sequences or signal sequences directing a protein to bind to a particular part of a cell
  • Insertional mutants typically involve the addition of material at a non-terminal point in the polypeptide This may include the insertion of an immunoreactive epitope or simply a single residue Terminal additions, called fusion proteins, are discussed below
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties ofthe polypeptide, such as stability against proteolytic cleavage, without the loss of other functions or properties
  • Substitutions of this kind preferably are conservative, that is, one amino acid is replaced with one of similar shape and charge
  • Conservative substitutions are well known in the art and include, for example, the changes of alanine to serine; arginine to lysine, asparagine to glutamine or histidine, aspartate to glutamate, cysteine to serine, glutamine to asparagine, glutamate to aspartate, glycine to proline, histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine, methionine to leucine or isoleucine; phenylalanine to tyrosine,
  • amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules
  • the hydropathic index of amino acids may be considered.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte & Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure ofthe resultant protein, which in turn defines the interaction ofthe protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte & Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8), tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5), glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those that are within ⁇ 1 are particularly preferred, and those within ⁇ 0 5 are even more particularly preferred
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include arginine and lysine, glutamate and aspartate, serine and threonine, glutamine and asparagine; and valine, leucine and isoleucine
  • peptide mimetics are peptide-containing molecules that mimic elements of protein secondary structure See, for example, Johnson et al, "Peptide Turn Mimetics” in BIOTECHNOLOGY AND PHARMACY, Pezzuto et al, Eds , Chapman and Hall, New York ( 1993)
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and antigen
  • a peptide mimetic is expected to permit molecular interactions similar to the natural molecule
  • fusion protein This molecule generally has all or a substantial portion of the native molecule, linked at the N- or C- terminus, to all or a portion of a second polypeptide.
  • fusions typically employ leader sequences from other species to permit the recombinant expression of a protein in a heterologous host.
  • Another useful fusion includes the addition of a immunologically active domain, such as an antibody epitope, to facilitate purification of the fusion protein Inclusion of a cleavage site at or near the fusion junction will facilitate removal of the extraneous polypeptide after purification
  • a immunologically active domain such as an antibody epitope
  • Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions
  • Protein purification techniques are well known to those of skill in the art These techniques involve, at one level, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic, immunologic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity)
  • Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis, isoelectric focusing
  • a particularly efficient method of purifying peptides is fast protein liquid chromatography or HPLC.
  • Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide
  • purified protein or peptide as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state
  • a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component ofthe composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more ofthe proteins in the composition.
  • purified to homogeneity is used to mean that the composition has been purified such that there is single protein species based on the particular test of purity employed for example SDS-PAGE or HPLC.
  • a preferred method for assessing the purity of a fraction is to calculate the specific protease activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number.”
  • the actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close- packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution ofthe sample.
  • Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix.
  • the column material is then able to specifically adsorb the substance from the solution.
  • Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below
  • the present invention also describes smaller FR-MP-related peptides for use in various embodiments of the present invention Because of their relatively small size, the peptides ofthe invention can also be synthesized in solution or on a solid support in accordance with conventional techniques Various automatic synthesizers are commercially available and can be used in accordance with known protocols See, for example, Stewart and Young, (1984), Tarn et al , (1983), Merrifield, (1986), and
  • the present invention also provides for the use of FR-MP proteins or peptides as antigens for the immunization of animals relating to the production of antibodies
  • FR-MP or portions thereof, will be coupled, bonded, bound, conjugated or chemically-linked to one or more agents via linkers, polylinkers or derivatized amino acids This may be performed such that a bispecific or multivalent composition or vaccine is produced
  • linkers, polylinkers or derivatized amino acids This may be performed such that a bispecific or multivalent composition or vaccine is produced
  • the methods used in the preparation of these compositions will be familiar to those of skill in the art and should be suitable for administration to animals, i.e., pharmaceutically acceptable Preferred agents as carriers are keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA) G.
  • KLH keyhole limpet hemocyannin
  • BSA bovine serum albumin
  • FR-MP was, in fact, a protease and not a GPI-specific phospholipase
  • an in vitro as was developed that permits the assaying of FR-MP activity
  • This assay may be employed in a number of different embodiments, for example, in assays for the diagnosis of disease states associated with abnormal expression of FR-MP or in the screening for inhibitors or stimulators of FR-MP synthesis, activity and/or stability
  • [ 3 H]leucine would be biosynthetically-incorporated proportionately to its distribution in the nascent polypeptide, i.e , 23% in the signal peptide, 36% in the C-terminal domain and 41% in other regions ofthe polypeptide
  • the C-terminal domain is hydrophobic, if EDTA-sensitive endoproteolytic cleavage occurred either within or proximal to this region, the PFR polypeptide would be expected to be converted to a relatively hydrophilic form with loss of specific radioactivity in the major fragment by up to 36% of the original value In fact, the metalloprotease did endoproteolytically alter the substrate in an EDTA-sensitive manner, and the net recovered radioactivity was -30% less than the original substrate
  • the metalloprotease in its substantially homogeneous form provides access to protein sequencing and antibodies, which in turn provides access to DNAs encoding the metalloprotease
  • DNA sequences deduced from the amino acid sequence of the metalloprotease can be prepared using conventional techniques, and used as probes to recover corresponding DNA's from genomic or cDNA libraries containing the metalloprotease DNA, or DNAs encoding similar metalloproteases having activity for cleaving hydrophobic FR to hydrophilic FR
  • Particular libraries include a human placental cDNA library and a HeLa (cervical cancer cell) cDNA library such as are available from Clonetech Laboratories, Inc , Palo Alto, CA
  • the purified metalloprotease can be used to raise antibodies which can be used to probe such libraries for DNA's encoding the metalloprotease Following cloning, such DNA's can then be incorporated in appropriate expression vectors and used to transform host cells (e.g.,
  • the present invention is not limited in scope to the human FR-MP gene, however, as one of ordinary skill in the art could, using these nucleic acids, readily identify related homologs in various other species (e.g., rat, rabbit, monkey, dog, mouse, gibbon, chimp, ape, baboon, cow, pig, horse, sheep, cat and other species)
  • any reference to a nucleic acid should be read as encompassing a host cell containing that nucleic acid and, in some cases, capable of expressing the product of that nucleic acid.
  • cells expressing nucleic acids of the present invention may prove useful in the context of screening for agents that induce, repress, inhibit, augment, interfere with, block, abrogate, stimulate or enhance the function of FR-MP.
  • Nucleic acids according to the present invention may encode an entire gene, a domain of FR-MP that expresses a protease function, or any other fragment ofthe FR-MP coding, non-coding or regulatory sequences.
  • the nucleic acid may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism
  • the nucleic acid would comprise complementary DNA (cDNA)
  • cDNA complementary DNA
  • mini-genes engineered molecules are sometime referred to as "mini-genes"
  • these and other nucleic acids of the present invention may be used as molecular weight standards in, for example, gel electrophoresis.
  • cDNA is intended to refer to DNA prepared using messenger RNA
  • mRNA as template.
  • DNA polymerized from a genomic, non- or partially-processed RNA template is that the cDNA primarily contains coding sequences of the corresponding protein There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy
  • FR-MP gene from a given species may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein (see Table 1 below)
  • an isolated nucleic acid encoding a FR-MP refers to a nucleic acid molecule that has been isolated free of total cellular nucleic acid
  • the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine (Table 1, below), and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages
  • sequences that have at least about ⁇ 0%, usually at least about 60%, more usually about 70%, most usually about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotides of a FR-MP gene will be sequences that encompassed by the present invention.
  • Nucleic acid sequences ofthe present invention may also be functionally defined as sequences that are capable of hybridizing to a nucleic acid segment encoding a FR-MP.
  • the DNA segments of the present invention include those encoding biologically functional equivalent FR-MP proteins and peptides, as described above
  • Functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged, or as a result of natural selection Changes designed by man may be introduced through the application of site-directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below
  • the present invention also encompasses DNA segments that are complementary, or essentially complementary, to a sequence encoding a FR-MP Nucleic acid sequences that are "complementary" are those that are capable of base-pairing according to the standard Watson-Crick complementary rules As used herein, the term
  • complementary sequences means nucleic acid sequences that are substantially complementary, as may be assessed by the same nucleotide comparison set forth above, or as defined as being capable of hybridizing to a nucleic acid segment encoding a FR-MP under relatively stringent conditions such as those described herein
  • the hybridizing segments may be shorter oligonucleotides Sequences of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length It is contemplated that exemplary oligonucleotides of 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more base pairs will be used, although others are contemplated Longer polynucleotides encoding 250, 500, 1000, 1212, 1500, 2000, 2500, 3000 or 3431 bases and longer are contemplated as well Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8 3), 75 mM KCl, 3 mM MgCl 2 , 10 mM dithiothreitol, at temperatures between approximately 20°C to about 37°C.
  • Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8 3), 50 mM KCl, 1 5 ⁇ M MgCl 2 , at temperatures ranging from approximately 40°C to about 72°C Formamide and SDS also may be used to alter the hybridization conditions
  • One method of using probes and primers of the present invention is in the search for genes related to FR-MP genes or, more particularly, homologs of FR-MP from non ⁇ human species Normally, the target DNA will be a genomic or cDNA library, although screening may involve analysis of RNA molecules By varying the stringency of hybridization, and the region of the probe, different degrees of homology may be discovered
  • Site-specific mutagenesis is a technique useful in the preparation of individual peptides, or biologically functional equivalent proteins or peptides, through specific mutagenesis of the underlying DNA
  • the technique further provides a ready ability to prepare and test sequence variants, incorporating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction ofthe sequence being altered.
  • the technique typically employs a bacteriophage vector that exists in both a single-stranded and double-stranded form.
  • Typical vectors useful in site-directed mutagenesis include vectors such as the Ml 3 phage These phage vectors are commercially available and their use is generally well known to those skilled in the art Double-stranded plasmids are also routinely employed in site-directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to a plasmid
  • site-directed mutagenesis is performed by first obtaining a single- stranded vector, or melting of two strands of a double-stranded vector which includes within its sequence a DNA sequence encoding the desired protein.
  • An oligonucleotide primer bearing the desired mutated sequence is synthetically prepared.
  • This primer is then annealed with the single-stranded DNA preparation, taking into account the degree of mismatch when selecting hybridization conditions, and subjected to DNA polymerizing enzymes such as E. coli polymerase I Klenow fragment, in order to complete the synthesis ofthe mutation-bearing strand.
  • E. coli polymerase I Klenow fragment DNA polymerizing enzymes
  • a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.
  • sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained
  • recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants
  • mutant proteases may not be non-functional. Rather, they may have aberrant functions that cannot be overcome by replacement gene therapy, even where the "wild-type” molecule is expressed in amounts in excess of the mutant polypeptide. Antisense treatments are one way of addressing this situation Antisense technology also may be used to "knock-out" function of FR-MP in the development of cell lines or transgenic mice for research, diagnostic and screening purposes.
  • Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences
  • polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA.
  • Inclusion of less common bases such as inosine, 5- methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
  • Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
  • Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject
  • Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron-exon splice junctions Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof The amount of exonic material included will vary depending on the particular exon and intron sequences used One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected
  • complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches Other sequences with lower degrees of homology also are contemplated For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme, see below) could be designed These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et al, 1987; Forster and Symons, 1987).
  • ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cook et al, 1981 ; Michel and Westhof,
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et al, 1981).
  • U.S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyme- mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991 ; Sarver et al, 1990).
  • ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.
  • expression vectors are employed to express a FR- MP polypeptide product, which can then be purified and, for example, be used to vaccinate animals to generate antisera or monoclonal antibody with which further studies may be conducted.
  • the expression vectors are used in gene therapy.
  • Expression requires that appropriate signals be provided in the vectors, and which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells.
  • regulatory elements such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells
  • Elements designed to optimize messenger RNA stability and translatability in host cells also are defined
  • the conditions for the use of a number of dominant drug selection markers for establishing permanent, stable cell clones expressing the products are also provided, as is an element that links expression of the drug selection markers to expression ofthe polypeptide
  • expression construct is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed
  • the transcript may be translated into a protein, but it need not be
  • expression includes both transcription of a gene and translation of mRNA into a gene product
  • expression only includes transcription ofthe nucleic acid encoding a gene of interest
  • the nucleic acid encoding a gene product is under transcriptional control of a promoter
  • promoter refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene
  • under transcriptional control means that the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase initiation and expression ofthe gene
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II
  • Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units
  • At least one module in each promoter functions to position the start site for RNA synthesis
  • the best known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation
  • promoter elements regulate the frequency of transcriptional initiation Typically, these are located in the region 30-1 10 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream ofthe start site as well
  • the spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another
  • the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline Depending on the promoter, it appears that individual elements can function either cooperatively or independently to activate transcription
  • the particular promoter employed to control the expression of a nucleic acid sequence of interest is not believed to be important, so long as it is capable of direction the expression of the nucleic acid in the targeted cell
  • a promoter that is capable of being expressed in a human cell
  • such a promoter might include either a human or viral promoter
  • the human cytomegalovirus (CMV) immediate early gene promoter can be used to obtain high-level expression of the coding sequence of interest
  • CMV cytomegalovirus
  • the SV40 early promoter the Rous sarcoma virus long terminal repeat
  • rat insulin promoter and glyceraldehyde-3 -phosphate dehydrogenase
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.
  • a promoter By employing a promoter with well-known properties, the level and pattern of expression of the protein of interest following transfection or transformation can be optimized. Further, selection of a promoter that is regulated in response to specific physiologic signals can permit inducible expression ofthe gene product.
  • Tables 2 and 3 list several elements/promoters which may be employed, in the context ofthe present invention, to regulate the expression ofthe gene of interest. This list is not intended to be exhaustive of all the possible elements involved in the promotion of gene expression but, merely, to be exemplary thereof.
  • Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins.
  • enhancers The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.
  • any promoter/enhancer combination (as per the Eukaryotic Promoter Data Base EPDB) could also be used to drive expression of the gene Eukaryotic cells can support cytoplasmic transcription from certain bacterial promoters if the appropriate bacterial polymerase is provided, either as part of the delivery complex or as an additional genetic expression construct.
  • NCAM Neural Cell Adhesion Molecule
  • SAA Human Serum Amyloid A
  • MMTV mammary tumor Glucocorticoids virus
  • TPA Collagenase Phorbol Ester
  • Insulin E Box Glucose Where a cDNA insert is employed, one typically will desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript
  • a polyadenylation signal to effect proper polyadenylation of the gene transcript
  • the nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals
  • a terminator are also contemplated as an element of the expression cassette. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.
  • the cells contain nucleic acid constructs of the present invention
  • a cell may be identified in vitro or in vivo by including a marker in the expression construct.
  • markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
  • a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed.
  • Immunologic markers also can be employed The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.
  • IRES elements are used to create multigene, or polycistronic, messages.
  • IRES elements are able to bypass the ribosome scanning model of 5' methylated cap- dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988).
  • IRES elements from two members of the picanovirus family polio and encephalomyocarditis have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991).
  • IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.
  • Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.
  • the expression construct comprises a virus or engineered construct derived from a viral genome.
  • the first viruses used as gene vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986). These have a relatively low capacity for foreign DNA sequences and have a restricted host spectrum. Furthermore, their oncogenic potential and cytopathic effects in permissive cells raise safety concerns. They can accommodate only up to 8 kB of foreign genetic material but can be readily introduced in a variety of cell lines and laboratory animals (Nicolas and Rubenstein, 1988; Temin, 1986).
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express an antisense polynucleotide that has been cloned therein In this context, expression does not require that the gene product be synthesized
  • the expression vector comprises a genetically engineered form of adenovirus
  • adenovirus a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992)
  • retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity
  • Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication
  • the El region (El A and E1B) encodes proteins responsible for the regulation of transcription ofthe viral genome and a few cellular genes
  • the expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990)
  • the products of the late genes including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP)
  • MLP major late promote
  • adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al, 1987), providing capacity for about 2 extra kB of DNA. Combined with the approximately
  • the maximum capacity of the current adenovirus vector is under 7.5 kB, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone and is the source of vector-borne cytotoxicity. Also, the replication deficiency of the El -deleted virus is incomplete. For example, leakage of viral gene expression has been observed with the currently available vectors at high multiplicities of infection (MOI) (Mulligan, 1993).
  • MOI multiplicities of infection
  • Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells As stated above, the preferred helper cell line is 293.
  • Racher et al (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.
  • natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium Following stirring at 40 ⁇ m, the cell viability is estimated with trypan blue.
  • Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows.
  • cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05 Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • the typical vector according to the present invention is replication-defective and will not have an adenovirus El region
  • the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
  • the polynucleotide encoding the gene of interest may also be inserted in lieu ofthe deleted E3 region in E3 replacement vectors as described by Karlsson et al, (1986) or in the E4 region where a helper cell line or helper virus complements the E4 defect.
  • Adenovirus is easy to grow and manipulate and exhibits broad host range in vitro and in vivo. This group of viruses can be obtained in high titers, e.g., 10 9 -10 n plaque-forming units per ml, and they are highly infective The life cycle of adenovirus does not require integration into the host cell genome The foreign genes delivered by adenovirus vectors are episomal and, therefore, have low genotoxicity to host cells
  • Adenovirus vectors have been used in eukaryotic gene expression (Levrero et al, 1991 , Gomez-Foix et al, 1992) and vaccine development (Grunhaus and Horwitz, 1992, Graham and Prevec, 1992) Recently, animal studies suggested that recombinant adenovirus could be used for gene therapy (Stratford-Perricaudet and Perricaudet, 1991, Stratford-Perricaudet et al, 1990, Rich et al, 1993) Studies in administering recombinant adenovirus to different tissues include trachea instillation
  • the retroviruses are a group of single-stranded RNA viruses characterized by an ability to convert their RNA to double-stranded DNA in infected cells by a process of reverse-transcription (Coffin, 1990)
  • the resulting DNA then stably integrates into cellular chromosomes as a provirus and directs synthesis of viral proteins
  • the integration results in the retention of the viral gene sequences in the recipient cell and its descendants
  • the retroviral genome contains three genes, gag, pol, and env that code for capsid proteins, polymerase enzyme, and envelope components, respectively
  • a sequence found upstream from the gag gene contains a signal for packaging of the genome into virions.
  • LTR long terminal repeat
  • Retroviral vectors are able to infect a broad variety of cell types
  • integration and stable expression require the division of host cells (Paskind et al, 1975).
  • retrovirus vectors usually integrate into random sites in the cell genome. This can lead to insertional mutagenesis through the interruption of host genes or through the insertion of viral regulatory sequences that can interfere with the function of flanking genes (Varmus et al, 1981)
  • Another concern with the use of defective retrovirus vectors is the potential appearance of wild-type replication- competent virus in the packaging cells This can result from recombination events in which the intact- sequence from the recombinant virus inserts upstream from the gag, pol, env sequence integrated in the host cell genome
  • new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al, 1988, Hersdorffer et al, 1990)
  • viral vectors may be employed as expression constructs in the present invention
  • Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988,
  • AAV adeno-associated virus
  • the expression construct In order to effect expression of sense or antisense gene constructs, the expression construct must be delivered into a cell This delivery may be accomplished in vitro, as in laboratory procedures for transforming cells lines, or in vivo or ex vivo, as in the treatment of certain disease states
  • One mechanism for delivery is via viral infection where the expression construct is encapsidated in an infectious viral particle
  • Non-viral methods for the transfer of expression constructs into cultured mammalian cells include calcium phosphate precipitation (Graham and Van Der Eb, 1973, Chen and Okayama, 1987, Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986, Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982, Fraley et al,
  • the nucleic acid encoding the gene of interest may be positioned and expressed at different sites.
  • the nucleic acid encoding the gene may be stably integrated into the genome ofthe cell This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation)
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA
  • Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed
  • the expression construct may simply consist of naked recombinant DNA or plasmids Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well Dubensky et al (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product
  • the expression construct may be entrapped in a liposome
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium Multilamellar liposomes have multiple lipid layers separated by aqueous medium They form spontaneously when phospholipids are suspended in an excess of aqueous solution The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991) Also contemplated are lipofectamine-DNA complexes Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful Wong et al, (1980) demonstrated the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo,
  • the liposome may be complexed with a hemagglutinating virus (HVJ) This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989)
  • HVJ hemagglutinating virus
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991)
  • HMG-1 nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG- 1
  • expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention
  • a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a particular gene into cells.
  • receptor-mediated delivery vehicles These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because ofthe cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu, 1993)
  • Receptor-mediated gene targeting vehicles generally consist of two components a cell receptor-specific ligand and a DNA-binding agent Several ligands hwe been used for receptor-mediated gene transfer The most extensively cnaracterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990).
  • ASOR asialoorosomucoid
  • transferrin Wang et al, 1990.
  • the delivery vehicle may comprise a ligand and a liposome
  • a nucleic acid encoding a particular gene also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes.
  • epidermal growth factor may be used as the receptor for mediated delivery of a nucleic acid encoding a gene in many tumor cells that exhibit upregulation of EGF receptor Mannose can be used to target the mannose receptor on liver cells
  • antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties
  • gene transfer may more easily be performed under ex vivo conditions
  • Ex vivo gene therapy refers to the isolation of cells from an animal, the delivery of a nucleic acid into the cells in vitro, and then the return ofthe modified cells back into an animal This may involve the surgical removal of tissue/organs from an animal or the primary culture of cells and tissues
  • Cloning of the FR-MP DNAs human cells is yet another aspect of the present invention
  • a variety of different starting materials may be employed, but a preferred embodiment includes the cloning of cDNA from a human choriocarcinoma (JEG-3) cell cDNA library
  • JEG-3 human choriocarcinoma
  • Various embodiments may be employed to achieve this cloning
  • oligonucleotide probes synthesized on the basis of predicted codon usage in the 5 '-most coding region of FR-MP
  • the 5 '-sequence is predicted from the results of N-terminal sequence of FR-MP Probes may be used in a variety of different hybridization formats, including Northern and Southern
  • Another cloning approach involves the use of antibodies or antiserum specific for FR-MP
  • antibodies or antiserum is used to purify nascent FR-MP chains during translation, along with the attached ribosomal and mRNA structures Conversion of the RNA to cDNA, followed by cloning and sequencing, then is undertaken
  • Yet a third approach is "expression" cloning In this technique, an expression library is screened for PFR proteolytic activity Identification of clones which possess this activity provide likely candidates for isolation and sequencing
  • FR-MP has been associated with certain malignancies Therefore, assays for FR-MP and FR-MP activity may be employed as a diagnostic or prognostic indicator of cancer More specifically, point mutations, deletions, insertions or regulatory perturbations relating to FR-MP may cause cancer or promote cancer development, cause or promoter tumor progression at a primary site, and/or cause or promote metastasis Other phenomena associated with malignancy that may be affected by FR- MP expression include angiogenesis and tissue invasion
  • One embodiment of the instant invention comprises a method for detecting variation in the expression of FR-MP This may comprise determining that level of FR-MP or determining specific alterations in the expressed product Obviously, this sort of assay has importance in the diagnosis of related cancers
  • Such cancer may involve cancers of the brain (glioblastomas, medulloblastoma, astrocytoma, oligodendroglioma, ependymomas), lung, liver, spleen, kidney, pancreas, small intestine, blood cells, lymph node, colon, breast, endometrium, stomach, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue
  • the compositions of the present invention may also be useful in the treatment of certain parasitic infections such as, for example, leishmaniasis.
  • the biological sample can be any tissue or fluid
  • Various embodiments include cells of the skin, muscle, facia, brain, prostate, breast, endometrium, lung, head & neck, pancreas, small intestine, blood cells, liver, testes, ovaries, colon, skin, stomach, esophagus, spleen, lymph node, bone marrow or kidney
  • Other embodiments include fluid samples such as peripheral blood, lymph fluid, ascites, serous fluid, pleural effusion, sputum, cerebrospinal fluid, lacrimal fluid, stool or urine
  • Nucleic acid used is isolated from cells contained in the biological sample, according to standard methodologies (Sambrook et al, 1989)
  • the nucleic acid may be genomic DNA or fractionated or whole cell RNA Where RNA is used, it may be desired to convert the RNA to a complementary DNA
  • the RNA is whole cell RNA, in another, it is poly-A RNA Normally, the nucleic acid is amplified
  • the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification
  • the identified product is detected
  • the detection may be performed by visual means (e.g., ethidium bromide staining of a gel)
  • the detection may involve indirect identification of the product via chemiluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Bellus, 1994)
  • alterations should be read as including deletions, insertions, point mutations and duplications
  • Point mutations result in stop codons, frameshift mutations or amino acid substitutions
  • Somatic mutations are those occurring in non-germline tissues
  • Germ-line tissue can occur in any tissue and are inherited Mutations in and outside the coding region also may affect the amount of FR-MP produced, both by altering the transcription ofthe gene or in destabilizing or altering the processing of either the transcript (mRNA) or protein
  • FISH fluorescent in situ hybridization
  • PFGE analysis direct DNA sequencing
  • Southern or Northern blotting single-stranded conformation analysis (SSCA)
  • RNAse protection assay allele-specific oligonucleotide (ASO)
  • ASO allele-specific oligonucleotide
  • dot blot analysis denaturing gradient gel electrophoresis
  • RFLP RFLP
  • primer as defined herein, is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process
  • primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed
  • Primers may be provided in double- stranded or single-stranded form, although the single-stranded form is prefe ⁇ ed Probes are defined differently, although they may act as primers Probes, while perhaps capable of priming, are designed to binding to the target DNA or RNA and need not be used in an amplification process
  • the probes or primers are labeled with radioactive species ( 32 P, 14 C, 35 S, 3 H, or other label), with a fluorophore (rhodamine, fluorescein) or a chemilluminescent (luciferase)
  • radioactive species 32 P, 14 C, 35 S, 3 H, or other label
  • fluorophore rhodamine, fluorescein
  • luciferase chemilluminescent
  • PCRTM polymerase chain reaction
  • PCR two primer sequences are prepared that are complementary to regions on opposite complementary strands of the marker sequence
  • An excess of deoxynucleoside triphosphates are added to a reaction mixture along with a DNA polymerase, e.g., Taq polymerase
  • a DNA polymerase e.g., Taq polymerase
  • the pnmers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified
  • Methods of reverse transcribing RNA into cDNA are well known and described in Sambrook et al, 1989
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases These methods are described in WO 90/07641 filed December 21, 1990 Polymerase chain reaction methodologies are well known in the art
  • LCR ligase chain reaction
  • SDA Strand Displacement Amplification
  • nucleic acid amplification procedures include transcription-based amplification systems (TAS), including nucleic acid sequence based amplification (NASBA) and 3SR (Kwoh et al, 1989, Gingeras et al, PCT Application WO 88/10315, inco ⁇ orated herein by reference in their entirety) Davey et al, EPO No 329 822 (inco ⁇ orated herein by reference in its entirety) disclose a nucleic acid amplification process involving cyclically synthesizing single- stranded RNA ("ssRNA”), ssRNA
  • Southern/Northern Blotting Blotting techniques are well known to those of skill in the art Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species
  • a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose
  • the different species should be spatially separated to facilitate analysis This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter
  • the blotted target is incubated with a probe (usually labeled) under conditions that promote denaturation and rehybridization. Because the probe is designed to base pair with the target, the probe will binding a portion of the target sequence under renaturing conditions. Unbound probe is then removed, and detection is accomplished as described above.
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al, 1989.
  • chromatographic techniques may be employed to effect separation.
  • chromatography There are many kinds of chromatography which may be used in the present invention: adso ⁇ tion, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography (Freifelder, 1982).
  • Products may be visualized in order to confirm amplification of the marker sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member ofthe binding pair carries a detectable moiety
  • detection is by a labeled probe
  • chromophore or radiolabel probes or primers identify the target during or following amplification
  • oligonucleotide primers may be designed to permit the amplification of sequences throughout an FR- MP gene that may then be analyzed by direct sequencing
  • Kit Components All the essential materials and reagents required for detecting and sequencing
  • kits This generally will comprise preselected primers and probes Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, SequenaseTM etc ), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification
  • enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, SequenaseTM etc ), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification
  • kits also generally will comprise, m suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe B.
  • Antibodies of the present invention can be used in characterizing the FR-MP content of healthy and diseased tissues, through techniques such as ELI S As and Western blotting This may provide a screen for the presence or absence of malignancy or as a predictor of future cancer
  • anti-FR-MP antibodies are immobilized onto a selected surface, preferably a surface exhibiting a protein affinity such as the wells of a polystyrene microtiter plate
  • a non-specific protein that is known to be antigenically neutral with regard to the test antisera such as bovine serum albumin (BSA), casein or solutions of powdered milk
  • the immobilizing surface After binding of antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the sample to be tested in a manner conducive to immune complex (antigen/antibody) formation.
  • the occurrence and even amount of immunocomplex formation may be determined by subjecting same to a second antibody having specificity for FR-MP that differs the first antibody
  • Appropriate conditions preferably include diluting the sample with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween ® These added agents also tend to assist in the reduction of nonspecific background
  • BSA bovine gamma globulin
  • PBS phosphate buffered saline
  • Tween ® phosphate buffered saline
  • the layered antisera is then allowed to incubate for from about 2 to about 4 hr, at temperatures preferably on the order of about 25° to about 27°C Following incubation, the antisera-contacted surface is washed so as to remove non-immunocomplexed material
  • a preferred washing procedure includes washing with a solution such as PBS/Tween ®
  • the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • one will desire to contact and incubate the second antibody-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hr at room temperature in a PBS- containing solution such as PBS/Tween ® ).
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g. , using a visible spectrum spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O , in the case of peroxidase as the enzyme label.
  • Quantitation is then achieved by measuring the degree of color generation, e.g. , using a visible spectrum spectrophotometer.
  • the preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with specificity for the primary antibody.
  • the antibody compositions of the present invention will find great use in immunoblot or Western blot analysis.
  • the antibodies may be used as high-affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof
  • a solid support matrix such as nitrocellulose, nylon or combinations thereof
  • immunoprecipitation followed by gel electrophoresis
  • these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background.
  • Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically-, radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard
  • Metalloprotease compositions of the invention may be used in therapeutic methods or studies
  • the metalloprotease may be used to cleave folate receptors from cells and thereby modulate the uptake of folates or other substances which are transported into the cell via the action ofthe folate receptor, e.g., substances such as methotrexate which are used in the treatment of neoplastic conditions or folate tethered liposomes bearing agents of biological import (chemotherapeutic agents, oligonucleotides, antisense constructs and the like)
  • the metalloproteases may be used for pu ⁇ oses related to those for which a range of antifolates are used and/or to study the action of such antifolates.
  • the metalloproteases and the antibodies specific to them may be used in the treatment of cancer.
  • the metalloproteases of the invention also may be used in the treatment or study of autoimmune diseases for example, rheumatoid arthritis and similar arthritic conditions.
  • FR 5-methyl-tetrahydrofolate and antifolates (such as methotrexate) with comparable rates to cells expressing only the reduced-folate carrier (Spinella et al, 1995).
  • FR have both physiologic and pharmacological importance. This leads to the possibility that overexpression of FR, and inhibition of FR-MP by antisense MP or antibodies to MP which will increase FR expression, will render cells more susceptible to antifolates While not a universal phenomenon, FR cDNA-transduced HeLa-IUi cells were analyzed for methotrexate-resistance, it was found that these cells were resistant to this antifolate
  • FR-MP modulation of the activity of FR-MP on cancer cells by a variety of biochemical (reconstitution of purified FR-MP), immunological (anti-FR-MP IgG to inhibit FR-MP) and molecular methods (transduction of antisense FR-MP cDNA) to effect an alteration on cell surface FR expression and cellular antifolate uptake or uptake of folate-tethered liposomes bearing cytotoxic agents is contemplated.
  • an 18-mer antisense molecule has been shown to effect synthesis of FR- ⁇ also has proved to be effective at inhibiting FR expression
  • compositions and routess of Administration Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions - expression vectors, oligonucleotides virus stocks, proteins, antibodies and drugs - in a form appropriate for the intended application Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals
  • compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium
  • a pharmaceutically acceptable carrier or aqueous medium
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like The use of such media and agents for pharmaceutically active substances is well know in the art
  • compositions of the present invention may include classic pharmaceutical preparations Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route This includes oral, nasal, buccal, rectal, vaginal or topical Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection Such compositions would normally be administered as pharmaceutically acceptable compositions, described supra
  • the active compounds may also be administered parenterally or intraperitoneally.
  • Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms
  • a surfactant such as hydroxypropylcellulose Dispersions
  • glycerol liquid polyethylene glycols, and mixtures thereof and in oils
  • oils Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syring
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption ofthe injectable compositions can be brought about by the use in the compositions of agents delaying abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
  • the use of such media and agents for pharmaceutical active substances is well known in the art Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the polypeptides of the present invention may be inco ⁇ orated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution)
  • the active ingredient may be inco ⁇ orated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • Routes of administration may be selected from intravenous, intrarterial, intrabuccal, intraperitoneal, intramuscular, subcutaneous, oral, topical, rectal, vaginal, nasal and intraocular.
  • aqueous solutions For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences" 15th Edition, pages 1035-1038 and 1570-1580)
  • Some variation in dosage will necessarily occur depending on the condition of the subject being treated
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Bi
  • liposomal formulations are contemplated Liposomal encapsulation of pharmaceutical agents prolongs their half-lives when compared to conventional drug delivery systems Because larger quantities can be protectively packaged, this allow the opportunity for dose-intensity of agents so delivered to cells This would be particularly attractive in the chemotherapy of cervical cancer if there were mechanisms to specifically enhance the cellular targeting of such liposomes to these cells In principle, this has been accomplished using FR
  • FR-expressing cells with liposome-entrapped doxorubicin and antisense DNA against human epidermal growth factor receptors in KB cells has been demonstrated (Lee and Low, 1994, Lee and Low, 1995, Wang et al, 1995)
  • transduction of HeLa-IU, cells with FR genes should induce them to proliferate slower (Sun et al, 1995) and take up more folate-tethered liposomes containing lethal cargo such as doxorubicin via FR (Lee and Low, 1995)
  • This method will be employed in cells expressing antisense FR-MP cDNA
  • Such studies should be able to demonstrate that the cells containing inactivated FR-MP would exhibit a lower IC50 using these folate tethered liposomes bearing doxorubicin
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers.
  • Phospholipids are used for preparing the liposomes according to the present invention and can carry a net positive charge, a net negative charge or are neutral Dicetyl phosphate can be employed to confer a negative charge on the liposomes, and stearylamine can be used to confer a positive charge on the liposomes
  • Liposomes are characterized by a phospholipid bilayer membrane and an inner aqueous medium Multilamellar liposomes have multiple lipid layers separated by aqueous medium.
  • lipid-nucleic acid complexes such as lipofectamine-nucleic acid complexes
  • the liposome may be complexed with a hemagglutinating virus (HVJ) This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989)
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991)
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1
  • expression vectors have been successfully employed in transfer and expression of a polynucleotide in vitro and in vivo, then they are applicable for the present invention
  • a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase
  • Lipids suitable for use according to the present invention can be obtained from commercial sources For example, dimyristyl phosphatidylcholine ("DMDM)
  • Phospholipids from natural sources such as egg or soybean phosphatidylcholine, brain phosphatidic acid, brain or plant phosphatidylinositol, heart cardiolipin and plant or bacterial phosphatidylethanolamine are preferably not used as the primary phosphatide, i.e., constituting 50% or more of the total phosphatide composition, because of the instability and leakiness ofthe resulting liposomes
  • Liposomes used according to the present invention can be made by different methods The size of the liposomes varies depending on the method of synthesis
  • a liposome suspended in an aqueous solution is generally in the shape of a spherical vesicle, having one or more concentric layers of lipid bilayer molecules.
  • Each layer consists of a parallel array of molecules represented by the formula XY, wherein X is a hydrophilic moiety and Y is a hydrophobic moiety.
  • the concentric layers are arranged such that the hydrophilic moieties tend to remain in contact with an aqueous phase and the hydrophobic regions tend to self-associate
  • the lipid molecules will form a bilayer, known as a lamella, ofthe arrangement XY-YX
  • Liposomes within the scope ofthe present invention can be prepared in accordance with known laboratory techniques
  • liposomes are prepared by mixing liposomal lipids, in a solvent in a container, e.g, a glass, pear-shaped flask
  • a container e.g, a glass, pear-shaped flask
  • the container should have a volume ten-times greater than the volume of the expected suspension of liposomes Using a rotary evaporator, the solvent is removed at approximately 40°C under negative pressure The solvent normally is removed within about 5 min to 2 hours, depending on the desired volume of the liposomes
  • the composition can be dried further in a desiccator under vacuum
  • the dried lipids generally are discarded after about 1 week because of a tendency to deteriorate with time
  • Dried lipids can be hydrated at approximately 25-50 mM phospholipid in sterile, pyrogen-free water by shaking until all the lipid film is resuspended The aqueous liposomes can be then separated into aliquots, each placed in a vial, lyophilized and sealed under vacuum
  • liposomes can be prepared in accordance with other known laboratory procedures the method of Bangham et al (1965), the contents of which are inco ⁇ orated herein by reference, the method of Gregoriadis, as described in DRUG CARRIERS IN BIOLOGY AND MEDICINE, G Gregoriadis ed (1979) pp 287-341, the contents of which are inco ⁇ orated herein by reference, the method of Deamer and Uster (1983), the contents of which are inco ⁇ orated by reference, and the reverse-phase evaporation method as described by Szoka and Papahadjopoulos (1978)
  • the aforementioned methods differ in their respective abilities to entrap aqueous material and their respective aqueous space-to-lipid ratios
  • the dried lipids or lyophilized liposomes prepared as described above may be reconstituted in a solution of nucleic acid and diluted to an appropriate concentration with an suitable solvent, e.g., DPBS The mixture is then vigorously shaken in a vortex mixer Unencapsulated nucleic acid is removed by centrifugation at 29,000 x g and the liposomal pellets washed The washed liposomes are resuspended at an appropriate total phospholipid concentration, e.g., about 50-200 mM The amount of nucleic acid encapsulated can be determined in accordance with standard methods After determination ofthe amount of nucleic acid encapsulated in the liposome preparation, the liposomes may be diluted to appropriate concentration and stored at 4°C until use
  • an appropriate solvent e.g., DPBS
  • Unencapsulated nucleic acid is removed by centrifugation at 29,000 x g and the liposomal pellets washed
  • the lipid dioleoylphosphatidylchoine is employed
  • Nuclease-resistant oligonucleotides were mixed with lipids in the presence of excess t-butanol The mixture was vortexed before being frozen in an acetone/dry ice bath The frozen mixture was lyophilized and hydrated with Hepes-buffered saline (1 mM Hepes, 10 mM NaCl, pH 7.5) overnight, and then the liposomes were sonicated in a bath type sonicator for 10 to 15 min.
  • the size of the liposomal-oligonucleotides typically ranged between 200-300 nm in diameter as determined by the submicron particle sizer autodilute model 370 (Nicomp, Santa Barbara, CA)
  • FR-MP proteolytic activity there are provided methods of screening compounds for activity against FR-MP proteolytic activity Such compounds may be useful in treatments where excessive FR-MP activity is involved
  • a preferred embodiment will be the adaptation of the in vitro activity assay using purified FR-MP, described elsewhere in this document At least 3 other assays may be employed, as discussed below
  • FR-MP transfect cells with an expression construct encoding FR-MP and contact cells with a putative inhibitor Monitoring of effects, both in the presence and absence of a candidate inhibitor, will provide a way of measuring the inhibitory effect of the substance
  • the examination of FR-MP will be on the basis of immunologic reactivity This can be accomplished in a variety of ways but, advantageously will be performed via radioimmune precipitation, Western blot or other such routine assay
  • the candidate inhibitor substance may be contacted with the cell directly
  • the candidate inhibitor substance may be reformulated to provide improved uptake
  • antisense oligonucleotides these may advantageously be formulated in liposomes or as virally-encapsulated expression vehicles.
  • polypeptides are to be tested, it may be advantageous to provide expression vectors encoding these molecules rather than the polypeptides themselves.
  • Effective amount for the purposes of the screening assay, is intended to mean an amount that will cause a detectable difference, and preferably a significant difference, in the measured effect as compared to a similar treatment without the candidate inhibitor substance.
  • the evaluation of the effects of the inhibitor may be undertaken.
  • 96-well trays may be employed in which several wells are reserved for controls while the remainder comprise test substances, usually with each substance being tested at several different amounts.
  • the formats are essentially as set forth above in screening for candidate inhibitors.
  • the present invention contemplates an antibody that is immunoreactive with a FR-MP molecule of the present invention, or any portion thereof.
  • An antibody can be a polyclonal or a monoclonal antibody. In a preferred embodiment, an antibody is a monoclonal antibody.
  • Means for preparing and characterizing antibodies are well known in the art (see, e.g., Howell and Lane, 1988).
  • a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal.
  • an immunogen comprising a polypeptide of the present invention
  • a wide range of animal species can be used for the production of antisera
  • an animal used for production of anti-antisera is a non-human animal including rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • Antibodies both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
  • a composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds ofthe present invention.
  • Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
  • the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to FR-MP-related antigen epitopes. Additionally, it is proposed that monoclonal antibodies specific to the particular FR-MP of different species may be utilized in other useful applications.
  • both polyclonal and monoclonal antibodies against FR-MP may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding other FR-MP. They may also be used in inhibition studies to analyze the effects of FR-MP-related peptides in cells or animals. Anti-FR-MP antibodies will also be useful in immunolocalization studies to analyze the distribution of FR-MP during various cellular events, for example, to determine the cellular or tissue-specific distribution of FR-MP polypeptides under different points in the cell cycle. A particularly useful application of such antibodies is in purifying native or recombinant FR-MP, for example, using an antibody affinity column.
  • a given composition may vary in its immunogenicity It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier
  • exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA)
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, /w-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization A variety of routes can be used to administer the immunogen
  • polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, inco ⁇ orated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified, partially purified or homogeneous FR-MP protein, polypeptide or peptide or cell expressing high levels of FR-MP.
  • a selected immunogen composition e.g., a purified, partially purified or homogeneous FR-MP protein, polypeptide or peptide or cell expressing high levels of FR-MP.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol.
  • B-cells B-lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 x IO 7 to 2 x 10 8 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas). Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986, Campbell, 1984).
  • the immunized animal is a mouse
  • P3-X63/Ag8.653, NSl/l .Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC1 1-X45-GTG 1.7 and S194/5XX0 Bul for rats, one may use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266,
  • GM1500-GRG2 LICR-LON-HMy2 and UC729-6 are all useful in connection with cell fusions.
  • Methods for generating hybrids of antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a
  • Fusion procedures usually produce viable hybrids at low frequencies, around 1 x IO "6 to 1 x 10 "8 . However, this does not pose a problem, as the viable, fused hybrids are differentiated from the parental, unfused cells (particularly the unfused myeloma cells that would normally continue to divide indefinitely) by culturing in a selective medium.
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • Exemplary and preferred agents are aminopterin, methotrexate, and azaserine Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity.
  • the assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.
  • the selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs.
  • the cell lines may be exploited for mAb production in two basic ways.
  • a sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion.
  • the injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid.
  • the body fluids of the animal such as serum or ascites fluid, can then be tapped to provide mAbs in high concentration.
  • the individual cell lines could also be cultured in vitro, where the mAbs are naturally secreted into the culture medium from which they can be readily obtained in high concentrations.
  • mAbs produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • FR folate receptor
  • PFR placental folate receptor
  • GPI glycosyl-phosphatidylinositol
  • HPLC HPLC
  • A Concanavalin A
  • TFA trifluoroacetic acid
  • FACS Fluorescence activated cell sorting.
  • the detergent-rich phase (250 ml) was diluted with 2 volumes of 10 mM Tris-HCl, pH 7 4, containing 500 mM NaCl and 1 mM MnCl 2 Con A-Sepharose, -15 ml of packed beads, which were pre-equilibrated in 10 mM Tris-HCl, pH 7 4, containing 500 mM NaCl, 0 5% Triton X-l 14, 1 mM CaCl 2 , and 1 mM MnCl 2 (Con A-binding buffer) (Ziegelbauer and Overath, 1992), were added to the sample and the mixture was incubated at 4°C for 16 h The beads were recovered by centrifugation at 1 OOOg for 10 min and washed once with 25 volumes of 10 mM Tris-HCl, pH 7 4, and twice for 10 min with 50 volumes of Con A-binding buffer The Con A-Sepharose-bound proteins were then batch-eluted over 16 h at 4°C with
  • Verification of Hydrophobicity of Purified Metalloenzyme The lyophilized HPLC-purified 63 kD metalloenzyme was resuspended in water and analyzed as follows One aliquot was subjected to temperature-induced phase-separation, and after the aqueous and micellar phase fractions were brought up to the same volume and final
  • Triton X-l 14 concentration samples derived from these phases were assayed for metalloenzyme activity Another aliquot was iodinated, and after removal of uninco ⁇ orated [ 125 I], Triton X-l 14 was added to a final concentration of 5% Following 3 cycles of temperature-induced phase-separation, with separate pooling of all 3 supernatants and all 3 pellets, aliquots of these pooled fractions were counted for radioactivity
  • Phenylthiocarbamyl amino acids were analyzed on an Applied Biosystems 130A Separation System using a 2 1 x 220 mm Brownlee C ⁇ 8 column (Foster City, CA) Phenylthiocarbamyl amino acid peaks were calibrated with the Pierce Amino acid Standard H
  • the Sp6/T7 transcription kit (Promega, Madison, WI) was used to generate the PFR mRNA Nascent PFR polypeptide was labeled with [ 3 H]leucine during in vitro translation of PFR RNA in the absence of microsomes Briefly, 2 ⁇ g of PFR mRNA was translated in vitro in the presence of 8 ⁇ Ci of [ 3 H]leucine, and 40 ⁇ l reticulocyte lysate (Sambrook et al, 1989) [ 3 H]leucine-labeled nascent PFR polypeptide was analyzed as follows one aliquot (20,000 cpm) was incubated with 50 ⁇ g of metalloenzyme (Con A-Sepharose eluate) in the absence or presence of 60 mM EDTA in a final volume of 220 ⁇ l for 16 h at 37°C The samples were subsequently loaded onto the reverse-phase HPLC (Vydac) column which was equilibrated with 95% buffer A (0.
  • FACS Fluorescence Activated Cell Sorting
  • HPLC-purified metalloprotease we did not investigate the basis for these observations. It is possible that the harsh reverse-phase HPLC conditions modified the metalloenzyme and led to protein-protein interactions with the substrate under conditions of gel filtration Finally, the apparently homogeneous protein was hydrophobic since -80% of HPLC-purified metalloenzyme and 87% of 125 I- metalloenzyme was recovered in the micellar phase following temperature-induced phase-separation analysis.
  • FIG 3 A shows the conversion of 125 I-hydrophobic
  • Table II shows the number of amino acid residues per mole of HPLC-purified metalloenzyme (based on 63,000 M r ) There were a total of 483 amino acid residues (minus tyrosine residues) which predicted a total M r of 59,179, comparable with that of deglycosylated metalloenzyme
  • the amino acids which were hydrophobic, hydrophilic and with ionizable side chains constituted 57%, 17%, and 26% ofthe protein, respectively
  • Fluorescence microscopy also demonstrated linear fluorescence exhibited by reaction of either anti-metalloprotease antiserum or anti-PFR antiserum with proteins on the plasma membranes of HeLa-IUi cells.
  • Normal human low density mononuclear cells enriched for hematopoietic progenitors are also known to express FR (Antony et al, 1991, Antony et al, 1987) When tested by FACS, nonimmune serum gave a mean channel fluorescence intensity value of 2
  • anti-PFR antiserum and anti-metalloprotease antiserum gave values of 81 and 30 units, respectively
  • both cultured human cervical carcinoma cells and normal human hematopoietic progenitor cells co-expressed cross-reacting moieties to PFR and placental metalloprotease on cell membranes
  • Anti-FR-MP Inhibits Protease Function It has been determined that anti-FR- MP antiserum specifically inhibits the protease activity in the Triton X-l 14 phase-separation assay system in 96-well plates (Yang et al, 1996) In these studies, the dose-response format involved the addition of increasing concentrations of anti-FR-MP antibodies to fixed amounts of FR-MP that cleaved fixed amounts of radiolabeled FR While there was no effect with non-immune serum, there was a dose-dependent reduction of FR-MP activity with increasing anti-FR-MP antiserum
  • anti-FR-MP antibodies are capable of blocking the activity of solubilized FR- MP, there is a high likelihood that they will also be functional in blocking the activity ofthe FR-MP in vivo
  • HeLa-IUj-LF Stable Adaptation to Low-Extracellular Folate Concentration
  • Rate of FR synthesis (pmol*/mg/h) 0.36 2.17
  • Cervical carcinoma is an acquired immunodeficiency syndrome (AIDS)-defining illness
  • AIDS acquired immunodeficiency syndrome
  • FR in cervical carcinoma (HeLa-IUi) cells can be up- or down-modulated by transduction of FR cDNA in the sense and antisense orientation (sense and antisense cells, respectively)
  • sense cells proliferated slower than antisense or untransduced cells, [methyl- 3 H]thymidine inco ⁇ oration into
  • TK thymidine kinase
  • TK activity conferred significant biological properties to sense cells (but not antisense or untransduced cells) as documented by augmented phosphorylation of 3 '-azido-3 '-deoxythymidine (AZT) and concomitantly greater sensitivity to the cytotoxic effects of AZT Conversely, sense cells were resistant to 5-fluorouracil and methotrexate, and methotrexate resistance was reversed by combination with AZT.
  • FR expression and TK activity indicates a previously unrecognized consequence of FR overexpression that warrants further investigation to identify the pathway linking FR and TK.
  • Antisense cells 34 1 ⁇ 7.3 180 0 ⁇ 17 5 1 1 ⁇ 0 1
  • MTX inhibits dihydrofolate reductase leading to a reduction in functional intracellular folates that participate in one-carbon metabolism, thereby inhibiting thymidylate synthase (Chello et al, 1976). It was hypothesized that sense cells should be more resistant to MTX through thymidylate salvage capacity (via TK). When the viability of cells cultured in 9 nM of 5-methyl-THF, 2.3 ⁇ M of folic acid, and increasing concentrations of MTX (5-10,000 nM) was determined, the IC 50 for antisense and untransduced cells was virtually identical at about 25 nM, whereas sense cells were markedly resistant to even 10,000 nM MTX. In addition, comparable results on resistance of sense cells to MTX were obtained using the assay of colony formation (Table 8). These data indicated that MTX resistance in sense cells could be due to the increased activity of TK.
  • FR mediate folate uptake into cells the independent role of FR in cell proliferation remains unclear.
  • the effects of transduction of FR cDNA, in sense or antisense orientation, on cervical carcinoma cells (which constitutively-overexpress FR genes) was measured with an adenoviral expression vector. It was determined that the integration of recombinant adeno-associated virions was not site-specific.
  • sense- and antisense-FR cDNA-transduced cells exhibited an increase and decrease in both FR mRNA and FR expression on the cell surface, respectively.
  • FR cDNA into cervical carcinoma cells modulated expression of FR and impacted on cell proliferation in vitro and in vivo.
  • FR- ⁇ cDNA were encapsidated in sense/anti sense orientation into AAV, transduced HeLa-IUi cells, and determined the functional consequences of over- and under-expression of FR on cell proliferation at high ( ⁇ M) extracellular folate concentration (Sun et al, 1995) This latter issue was important to eliminate the variable of intracellular folates on cell proliferation among various cohorts studied, since ⁇ M extracellular folate concentration ensured passive diffusion of folic acid into cells (Antony, 1992).
  • FR which mediate the cellular uptake of folates and antifolates are inversely regulated by the extracellular folate concentration.
  • cervical carcinoma (HeLa-IU i) cells which overexpress FR, up- and down-regulation of FR is primarily modulated at the translational level; accordingly, the potential for interaction of c/s-elements in FR- ⁇ mRNA and tr ⁇ ws-factors from these cells was determined Using gel-shift assays, two signals were identified which specifically derived from the interaction ofthe 5'-UTR of FR- ⁇ mRNA with cytosolic extracts from HeLa-IUi cells RNase Tl mapping revealed that both signals were from protein(s) interacting with two partially overlapping RNA sequences between nucleotides -133 to -116 and -158 to -116, upstream of the translation start site.
  • RNA of Tobacco Rinspot Virus Nature (London), 328 802-805, 1987 Ghosh & Bachhawat, "Targeting of liposomes to hepatocytes," In Liver diseases. targeted diagnosis and therapy using specific receptors and ligands. Wu & Wu
  • Nicolas & Rubenstein "Retroviral vectors," In Vectors. A survey of molecular cloning vectors and their uses. Rodriguez & Denhardt (eds ), Stoneham
  • Boiron eds ), Editions John Libbey Eurotext, France, 1991 Stratford-Perricaudet et al, "Evaluation ofthe transfer and expression in mice of an enzyme-encoding gene using a human adenovims vector," Hum. Gene Ther.,
  • Varmus et al "Retroviruses as mutagens Insertion and excision of a nontransforming provirus alter the expression of a resident transforming provirus," Cell, 25 23- 36, 1981

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Abstract

L'invention se rapporte à des préparations homogènes d'une métalloprotéase dirigée sur un récepteur de folate (FR-MP) et à des procédés d'obtention et d'utilisation de ces préparations. L'enzyme purifié, élué sous forme d'un seul pic de protéine en chromatographie liquide à hautes performances à phase inverse et par SDS-PAGE, a révélé une seule espèce de 63 000 Mr, qui a été réduite à 58 000 Mr par déglycosylation. L'invention concerne également le gène correspondant, des anticorps spécifiques de FR-MP et des procédés pour surveiller l'expression de FR-MP.
PCT/US1997/004595 1996-03-21 1997-03-21 Metalloproteases dirigees sur un recepteur de folate et leurs procedes d'utilisation WO1997034501A1 (fr)

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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF BIOLOGICAL CHEMISTRY, 05 February 1991, Volume 266, No. 4, ELWOOD et al., "The Conversion of the Human Membrane-Associated Folate Binding Protein (Folate Receptor) to the Soluble Folate Binding Protein by a Membrane-Associated Metalloprotease", pages 2346-2353. *
JOURNAL OF BIOLOGICAL CHEMISTRY, 05 July 1991, Volume 266, No. 19, VERMA et al., "Kinetic Analysis, Isolation and Characterization of Hydrophilic Folate-Binding Proteins Released from Chorionic Villi Cultured Under Serum-Free Conditions", pages 12522-12535. *
JOURNAL OF BIOLOGICAL CHEMISTRY, 10 May 1996, Volume 271, No. 19, YANG et al., "Isolation and Characterization of a Folate Receptor-Directed Metalloprotease from Human Placenta", pages 11493-11499. *
JOURNAL OF BIOLOGICAL CHEMISTRY, 25 February 1992, Volume 267, No. 6, VERMA et al., "Evidence that the Hydrophobicity of Isolated, in Situ and de Novo Synthesized Native Human Placental Folate Receptors is a Function of Glycosyl-Phosphatidylinositol Anchoring to Membranes", pages 4119-4127. *

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