DESCRIPTION

FOLATE RECEPTOR-DIRECTED METALLOPROTEASES AND METHODS FOR USING SAME

BACKGROUND OF THE INVENTION

A. Field of the Invention

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

B. Related Art

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) Conversely, 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

There are three distinct mechanisms for cellular folate transport (Antony, 1992) 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, μM) extracellular folate concentrations Cellular folate transport also can be mediated by

-38 kD membrane-associated folate receptors (FR) which bind physiologic folates (and antifolates) with high affinity in the nanomolar (nM) range (Antony, 1992, Antony, 1996) FR are expressed in rapidly proliferating normal cells, but are also overexpressed in several cancers including cervical carcinoma

There is no evidence that the reduced-folate carrier- and FR-mediated folate-transport systems communicate with one another (Dixon et ai, 1992) Significantly, recent studies indicate that although high affinity FR and the low affinity folate transporter (the reduced-folate carrier) function essentially independently of one another when enough FR is expressed, these proteins mediate the uptake of folates and methotrexate with comparable rates as cells expressing only the reduced-folate carrier (Spinella et ai, 1995) Together, these data predict that the components and mechanism(s) of regulation of FR expression have a significant impact on physiological cellular acquisition and transcellular transport of folates, as well as in chemotherapy of malignancies with antifolates Thus, perturbation of FR apparently does not lead to compensatory increase in folate uptake via other pathways Passive diffusion of folates into cells is operative only at high extracellular folate concentrations (Antony et al, 1989a), but it has been demonstrated that it is also operative in transplacental folate transport in combination with FR (Antony, 1996, Henderson et al, 1995)

Three human FR cDNA isoforms, FR-α, -β, and -γ (Antony, 1996) have been cloned Most studies related to transfection/transduction of FR cDN A into cells have used FR-α 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) 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)

Human placental FR (PFR) recently have been shown to have a major functional role as modulators of maternal-to-fetal transplacental folate transport (Henderson et al, 995) Native hydrophobic PFR are glycosyl-phosphatidylinositol (GPI)-anchored proteins, have a M _r of 35,000, and require detergent for solubilization out of membranes (Verma et al, 1992) Because of the GPI anchor, PFR are susceptible to cleavage to soluble forms by GPI-anchor specific phospholipases C and

D (Verma et al, 1992)

Through studies on detergent-solubilized crude placenta (containing cytosolic, nuclear and membrane proteins), a specific Mg ² " -dependent enzyme was identified which converted hydrophobic PFR to hydrophilic forms that retained ligand-binding capacity (Antony et al, 1989) The functional nature of this metalloenzyme in chorionic villi cultured under serum-free conditions has been demonstrated (Verma and

Antony, 1991), although in this earlier work only crude preparations were available Since hydrophilic and GPI-anchored FR from nasopharyngeal carcinoma (KB) cells (Luhrs and Slomiany, 1989) have similar amino-terminal amino acid sequences (Luhrs et al, 1987), the cleavage site for this placental metalloenzyme was provisionally assigned to the carboxyl (C)-terminal domain ofthe native PFR species (Antony, 1992, Verma and Antony, 1991)

Furthermore, isolation of 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). This lent further support to the conclusion that the placental metalloenzyme was a protease; however, such data were indirectly generated, and a direct demonstration of endoproteolytic cleavage of the native PFR by the metalloenzyme was lacking. In addition, all previous studies which demonstrated metalloenzyme activity relied on gel filtration in Triton X-100 which reliably separates the hydrophobic FR substrate (apparent 160 kD) from its hydrophilic product (40 kD) (Antony et al, 1989, Verma and Antony, 1991, Elwood et al, 1991). However, gel filtration is a cumbersome assay which is not easily adaptable to analysis of multiple variables required in isolation of proteins

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,

Triton X-100-solubiIized KB and placental membranes which converted [ ³ H]leucine- labeled hydrophobic FR to hydrophilic forms, providing a more direct indication that

this enzyme was a metalloprotease (as opposed to a GPI-specific phospholipase) 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) Thus, it is still unclear whether 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

Thus, there remains a need to further define the characteristics and specificity of the metalloprotease involved in PFR cleavage This calls for homogeneous preparations of FR-directed metalloproteases, antibodies to such metalloproteases, and to methods for using the same in functional assays

SUMMARY OF THE INVENTION

It is, therefore, a goal of the present invention to provide both 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

In a particular embodiment, 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 In certain aspects of the invention, the metalloenzyme-rich material is human placental tissue In particular embodiments, the human placental is homogenized, centrifuged to yield a supernatant which is separated therefrom The detergent may be a non-ionic detergent In preferred embodiments,

the non-ionic detergent is Triton X-l 14™ In certain aspects of the present invention, the metalloenzyme-detergent mixture is subjected to temperature-induced phase separation at 30°C

The isolating step may comprise affinity chromatography and concentration In particular aspects, 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

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.

In yet another aspect ofthe present invention, there is provided a purified folate receptor-directed metalloprotease (FR-MP) having the characteristics of a molecular weight of about 55,000 to about 63,000 daltons; activation by Mg ²⁺ , Mn ⁺ , Ca ²⁺ and

Zn ²⁺ ; and having the ability to convert hydrophobic placental folate receptor to its hydrophilic form.

Also contemplated by the present invention is a monoclonal antibody that reacts immunologically with a folate receptor-directed metalloprotease (FR-MP). The present invention further provides a polyclonal antiserum that reacts immunologically with a folate receptor-directed metalloprotease (FR-MP).

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. In other embodiments 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. In particular embodiments the determining comprises detergent-aqueous phase separation of PFR. In certain embodiments the detergent used is Triton X-l 14™. 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 In certain embodiments 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 In particular aspects, it is envisioned that the sample may be purified prior to the contacting In those aspects where the sample is purified, 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

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 In preferred aspects of the present invention, 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. In particular embodiments it is envisioned that the DNA may contain a coding region or a non-coding region or both. In other embodiments the DNA comprises intron-exon junction. In yet other embodiments the DNA comprises a transcription start site. The DNA also may comprise a translation start site or be part of an expression construct.

In another embodiment, 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.

In yet another embodiment, 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.

Other objects, features and advantages of the present invention will become apparent from the following detailed description It should be understood, however, that the detailed description and the specific examples, while indicating preferred

embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description

DESCRIPTION OF THE FIGURES

The following drawings form part ofthe present specification and are included to further demonstrate certain aspects of the present invention The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein

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 hydrophobic PFR for various times indicated at 37°C The results are the mean of triplicate studies and each value did not deviate more than 5% from the mean

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 μg 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 μg in 100 μl 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. 3C Determination of pH optimum for metalloenzyme activity in the absence and presence of 60 mM EDTA FIGS. 3D-F Characteristics of inhibition with 1,10-phenanthroline (FIG 3D), and reactivation of metalloenzyme (50 μg) 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

1.10 diluted (interrupted line) and undiluted (dotted line) anti-metalloprotease antiserum, respectively; 1 x IO ⁴ cells analyzed.

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.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention involves the isolation, characterization and use of a new metalloprotease, designated as folate receptor-metalloprotease or FR-MP. The existence of this molecule has been hypothesized for some years, but the present inventors have, for the first time, isolated FR-MP to apparent homogeneity on reverse phase HPLC. This major advance creates a variety of different exploitations that previously were not possible. 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⁺ .

Metalloprotease derived from human placental tissue, in substantially homogeneous form, was prepared using affinity chromatography and reverse phase HPLC as major purification steps. In a specific embodiment 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.

Several results indicated that the metalloprotease was hydrophobic, including the fact that the crude EDTA-sensitive metalloprotease was solubilized from placental membranes with Triton XI 14™ 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. Interestingly, however, when the placental metalloprotease was originally purified, it was recovered as a hydrophilic species. Subsequent analysis revealed that prolonged storage of the Con A-Sepharose elution for -1 week at 4°C (during multiple batch elutions) led to conversion of the initially hydrophobic metalloprotease to a hydrophilic species. Thus, the basis for the hydrophobicity of the metalloprotease must exist within either a lipid tail (possibly a GPI anchor), or a short hydrophobic polypeptide tail, and that the activity responsible for its conversion to a hydrophilic species resides in the Con A- Sepharose eluate.

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.

In the examples below, a rapid assay for FR-MP activity is described. Because

(i) the GPI-linked hydrophobic PFR sequesters in the micellar phase at the cloud point of Triton X-l 14 (Verma et al, 1992), (ii) the hydrophilic PFR product sequesters in

the aqueous phase, and (iii) the EDTA-sensitive enzyme in solubilized human placenta can completely cleave all native hydrophobic GPI-anchored FR into hydrophilic forms (Verma et al, 1992; Antony et al, 1989, Antony et al, 1981), it is a simple matter to determine if FR is cleaved and, hence, if FR-MP activity is present. This assay may be adapted for high through-put experiments with other proteases which convert a hydrophobic substrate to a hydrophilic product where multi-well tray formats are employed.

Other aspects of the present invention include a method for treating cells to remove folate receptors therefrom, which includes the step of contacting the cells with a construct that allows the expression of a metalloprotease so as to remove folate receptors from the cells and methods for screening for inhibitors or stimulators of FR- MP activity. Related compositions include genes encoding FR-MP and antibodies specific for an FR-MP.

The biological significance of the conversion of placental hydrophobic to hydrophilic FR remains under study. In the case of FR on malignant and normal cells, the conversion of hydrophobic FR to hydrophilic FR as mediated by a metalloprotease or GPI-specific phospholipase can potentially be an important mechanism for post- translational regulation ofthe expression of FR on the cell surface. Although a major role for the metalloprotease was not identified in the placenta at term (Henderson et al, 1995), its activity could easily determine the extent of acquisition of folates by FR on trophoblasts, thereby influencing placental growth and development. However, no probes (antibodies or cDNA) are available from this prior work to study such PFR- directed enzymes. In theory, inhibition of MP activity can potentially be exploited to increase FR on cell surfaces in experimental therapeutics of cancer cells which relies on increased FR to mediate cytotoxicity of these cells. Conversely, overexpression of MP activity in cancer cells which rely on FR for folate uptake can theoretically reduce FR expression thereby depleting cells of folate and thus leading to cell kill.

For the purpose of promoting an understanding of the principles of the invention, reference will now be made to certain preferred embodiments thereof and

specific language will be used to describe the same It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations, further modifications and applications of the principles of the invention as described herein being contemplated as would normally occur to one skilled in the art to which the invention relates

A. FR-MP Polypeptides and Protein Compositions

According to the present invention, 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

In addition to the entire molecule, 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

These 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)

A. Functional Aspects

When the present application refers to the function of an FR-MP or "wild-type" activity, it is meant that the molecule in question has the ability to cleave PFR from its insoluble (hydrophobic) to soluble (hydrophilic) form. Of course, it is understood that there may also be other substrates that have yet to be identified Determination of which molecules possess protease activity may be determined using assays familiar to

those of skill in the art For example, use of an PFR substrate permits assay of FR-MP activity, as discussed below

B. 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, leucine or methionine, serine to threonine; threonine to serine; tryptophan to tyrosine, tyrosine to tryptophan or phenylalanine, and valine to isoleucine or leucine

It may be possible, in fact, to improve certain ofthe properties of a FR-MP For example, certain 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

Since it is the interactive capacity and nature of a protein that defines that protein's

biological functional activity, certain amino acid substitutions can be made in a protein sequence, and its underlying DNA coding sequence, and nevertheless obtain a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the sequences of FR-MP without appreciable loss of their biological utility or activity, as discussed below, and perhaps with improved or enhanced activity or stability. Table 1 shows the codons that encode particular amino acids.

In making such changes, 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).

It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, ;. e. , still obtain a biological functionally equivalent protein.

In making such changes, the substitution of amino acids whose hydropathic indices are within ±2 is preferred, those which are within ±1 are particularly preferred, and those within ±0.5 are even more particularly preferred.

It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent 4,554,101, incorporated herein by reference, states that the greatest local average hydrophilicity of a protein, as

governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein As detailed in U S Patent 4,554,101, the following hydrophilicity values have been assigned to amino acid residues arginine (+3 0), lysine (+3 0), aspartate (+3 0 ± 1), glutamate (+3 0 ± 1), serine (+0 3), asparagine (+0 2), glutamine (+0.2), glycine (0), threonine (-0 4), proline (-0 5 ± 1), alanine (-0 5), histidine *-0.5), cysteine (-1 0), methionine (-1.3), valine (-1 5), leucine (-1 8), isoleucine (-1.8), tyrosine (-2 3), phenylalanine (-2 5), tryptophan (-3.4)

It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein. In such changes, the 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

As outlined above, 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

Another embodiment for the preparation of polypeptides according to the invention is the use of peptide mimetics 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 These principles may be used, in conjunction with the principles outline above, to engineer second generation molecules having many ofthe natural properties of FR-MP, but with altered and even improved characteristics

C. Fusion Proteins

A specialized kind of insertional variant is the 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. For example, 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 Other useful fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions

D. Purification of Proteins One of the embodiments of the present invention is the purification of FR-MP and fragments and variants thereof. 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

The term "purified protein or peptide" as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state A purified protein or

peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.

Generally, "purified" will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological 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. The term "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.

Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure.

These include, for example, determining the specific protease activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. 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.

There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low

pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al, 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.

High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain 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. 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

E. Synthetic Peptides 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

Barany and Merrifield (1979), each incorporated herein by reference Short peptide sequences, or libraries of overlapping peptides, usually from about 6 to about 50 amino acids, which correspond to the regions of an FR-MP, readily can be synthesized and then screened in screening assays designed to identify reactive peptides Alternatively, recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide ofthe invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression

F. Antigen Compositions

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 It is envisioned that either 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 It is further envisioned that 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. Assays for FR-MP A ctivity

Experiments were performed to demonstrate that isolated FR-MP was, in fact, a protease and not a GPI-specific phospholipase In so doing, 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

Synthesized [ ³ H]leucine-labeled nascent PFR substrate was generated in the absence of microsomes As pointed out above (Elwood et al, 1991), based on the deduced amino acid sequence from PFR cDNA (Elwood, 1989), there are 257 amino acid residues among which there are a total of 22 leucine residues - 5 are within the signal peptide (amino acids 1 to 25), 8 are in the hydrophobic C-terminal domain

(between amino acids 227 to 257), while the other 9 are distributed between amino acids 26 and 226 The nascent PFR polypeptide translated in vitro in the absence of microsomes would be full length and not be truncated in its hydrophobic C-terminal domain during addition of the GPI-anchor (Antony and Miller, 1994; Udenfriend and

Kodukula, 1995) Therefore, [ 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

Since 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

What remains unexplained is the paradoxical conversion of the [ ³ H]leucine- labeled nascent PFR polypeptide to a more hydrophobic species by the metalloprotease, a finding which would not have been predicted based on data generated with the mature protein However, three lines of evidence supported the

premise that the [ ³ H]leucine-labeled nascent PFR polypeptide had a different folded structure when compared to the native, mature, fully processed (glycosylated and GPI- anchored) PFR These included its relatively poorer immunoprecipitation by anti-PFR antiserum and its aberrant elution as an apparent -80 kD species on gel filtration in Triton X-l 00 In addition, it previously was observed that, following gentle methods of iodination, even mature (hydrophilic) PFR are extremely susceptible to unfolding with resultant exposure of core hydrophobic amino acids which interact with Triton X- 100 This led to major alterations in gel filtration profiles and reduced recognition by anti-PFR antiserum (Antony et al, 1987) similar to that noted with nascent PFR polypeptide Therefore, it is possible that subsequent to cleavage of the [TTJleucine- labeled nascent PFR polypeptide by the metalloprotease, the exposure to organic solvents during HPLC may have led to its further unfolding, leading to exposure and interaction of additional core hydrophobic regions with the hydrophobic HPLC column, as also noted previously (Zhou et al, 1990) This may explain why cleavage ofthe nascent PFR polypeptide versus mature PFR by the same metalloprotease led to the observed differences in the hydrophobic properties of the product Nonetheless, this assay presents a clear method for assessing FR-MP activity in vitro

B. Nulceic acids encoding FR-MP In addition, the metalloprotease in its substantially homogeneous form provides access to protein sequencing and antibodies, which in turn provides access to DNAs encoding the metalloprotease For example, 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 Likewise, 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., bacterial or mammalian cells), which can be cultured to form recombinant metalloprotease

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)

Similarly, 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. In addition to therapeutic considerations, 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.

A . Nucleic A cids Encoding 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. As stated above, the nucleic acid may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism

In preferred embodiments, however, the nucleic acid would comprise complementary DNA (cDNA) Also contemplated is a cDNA plus a natural intron or an intron derived from another gene, such engineered molecules are sometime referred to as "mini-genes " At a minimum, these and other nucleic acids of the present invention may be used as molecular weight standards in, for example, gel electrophoresis.

The term "cDNA" is intended to refer to DNA prepared using messenger RNA

(mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or

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

It also is contemplated that a given 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)

As used in this application, the term "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

TABLE 1

Amino Acids Codons

Alanine Ala A GCA GCC GCG GCU

Cysteine Cys C UGC UGU

Aspartic acid Asp D GAC GAU

Glutamic acid Glu E GAA GAG

Phenylalanine Phe F UUC uuu

Glycine Gly G GGA GGC GGG GGU

Histidine His H CAC CAU

Isoleucine He I AUA AUC AUU

Lysine Lys K AAA AAG

Leucine Leu L UUA UUG CUA CUC CUG CUU

Methionine Met M AUG

Asparagine Asn N AAC AAU

Proline Pro P CCA CCC CCG ecu

Glutamine Gin Q CAA CAG

Arginine Arg R AGA AGG CGA CGC CGG CGU

Serine Ser S AGC AGU UCA UCC UCG UCU

Threonine Thr T ACA ACC ACG ACU

Valine Val V GUA GUC GUG GUU

Tryptophan Trp W UGG

Tyrosine Tyr Y UAC UAU

Allowing for the degeneracy ofthe genetic code, sequences that have at least about ^ς 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

B. Oligonucleotide Probes and Primers

Naturally, 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

Alternatively, 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

Suitable hybridization conditions will be well known to those of skill in the art In certain applications, for example, substitution of amino acids by site-directed mutagenesis, it is appreciated that lower stringency conditions are required Under these conditions, hybridization may occur even though the sequences of probe and target strand are not perfectly complementary, but are mismatched at one or more positions Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature For example, a medium stringency condition could be provided by about 0 1 to 0 25 M NaCl at temperatures of about 37°C to about 55°C, while a low stringency condition could be provided by about 0 15 M to about 0 9 M salt, at temperatures ranging from about 20°C to about 55°C Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results

In other embodiments, 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 μM 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

Another way of exploiting probes and primers of the present invention is in site-directed, or site-specific mutagenesis 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. Typically, 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

In general, 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. Thus, 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.

The preparation of 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 For example, recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxylamine, to obtain sequence variants

C. Antisense Constructs In some cases, 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 By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (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.

Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide 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

As stated above, "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

It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence

D. Ribozymes

Another approach for addressing the "dominant negative" mutant proteases is through the use of ribozymes. Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromol ecules has emerged as useful in this endeavor. 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). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cook et al, 1981 ; Michel and Westhof,

1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") ofthe ribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et al, 1981). For example, U.S. Patent No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme- mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991 ; Sarver et al, 1990). Recently, it was reported that ribozymes elicited genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme.

E. Vectors for Cloning, Gene Transfer and Expression

Within certain embodiments 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. In other embodiments, 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 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

(i) Regulatory Elements Throughout this application, the term "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 In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product In other embodiments, expression only includes transcription ofthe nucleic acid encoding a gene of interest

In preferred embodiments, the nucleic acid encoding a gene product is under transcriptional control of a promoter A "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 The phrase "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

The term 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 These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of

approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins

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

Additional 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 In the TK promoter, 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 Thus, where a human cell is targeted, it is preferable to position the nucleic acid coding region adjacent to and under the control of a promoter that is capable of being expressed in a human cell Generally speaking, such a promoter might include either a human or viral promoter

In various embodiments, the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3 -phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest 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.

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.

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.

Below is a list of viral promoters, cellular promoters/enhancers and inducible promoters/enhancers that could be used in combination with the nucleic acid encoding a gene of interest in an expression construct (Table 2 and Table 3). Additionally, 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.

TABLE 2

ENHANCER/PROMOTER

Immunoglobulin Heavy Chain

Immunoglobulin Light Chain

T-Cell Receptor

HLA DQ α and DQ β

β-Interferon

Interleukin-2

Interleukin-2 Receptor

MHC Class 13 5

MHC Class II HLA-DRα

β-Actin

Muscle Creatine Kinase

Prealbumin (Transthyretin)

Elastase /

Metallothionein

Collagenase

Albumin Gene

α-Fetoprotein

τ-Globin

TABLE 2 CONTINUED β-Globin

e-fos

c-HA-ras

Insulin

Neural Cell Adhesion Molecule (NCAM)

αl-Antitrypsin

H2B (TH2B) Histone

Mouse or Type I Collagen

Glucose-Regulated Proteins (GRP94 and GRP78)

Rat Growth Hormone

Human Serum Amyloid A (SAA)

Troponin I (TN I)

Platelet-Derived Growth Factor

Duchenne Muscular Dystrophy

SV40

Polyoma

Retroviruses

Papilloma Virus

Hepatitis B Virus

Human Immunodeficiency Virus

Cytomegalovirus

Gibbon Ape Leukemia Virus -

TABLE 3

Element Inducer

MT Π Phorbol Ester (TPA) Heavy metals

MMTV (mouse mammary tumor Glucocorticoids virus)

β-Interferon poly(rI)X poly(rc)

Adenovirus 5 E2 Ela

c-jun Phorbol Ester (TPA), H ₂ O ₂

Collagenase Phorbol Ester (TPA)

Stromelysin Phorbol Ester (TPA), JJL-1

SV40 Phorbol Ester (TPA)

Murine MX Gene Interferon, Newcastle Disease Virus 1

GRP78 Gene A23187

α-2-Macroglobulin IL-6

Vimentin Serum

MHC Class I Gene H-2kB Interferon

HSP70 Ela, SV40 Large T Antigen

Proliferin Phorbol Ester-TPA

Tumor Necrosis Factor FMA

Thyroid Stimulating Hormone α Thyroid Hormone Gene

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 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 Also contemplated as an element of the expression cassette is a terminator These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

(ii) Selectable Markers

In certain embodiments of the invention, 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. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. Usually the inclusion of 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. Alternatively, 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.

(iii) Multigene Constructs and IRES

In certain embodiments of the invention, the use of internal ribosome binding sites (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.

(iv) Delivery of Expression Vectors There are a number of ways in which expression vectors may introduced into cells. In certain embodiments of the invention, the expression construct comprises a virus or engineered construct derived from a viral genome. The ability of certain viruses to enter cells via receptor-mediated endocytosis, to integrate into host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). 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).

One of the preferred methods for in vivo delivery involves the use of an adenovirus expression vector. "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

Knowledge ofthe genetic organization of adenovirus, a 36 kB, linear, double-stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kB (Grunhaus and Horwitz, 1992) In contrast to 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

Also, 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 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) The MLP, (located at 16 8 m.u ) is particularly efficient duπng the late phase of infection, and all the mRNA's issued from this promoter possess a 5 '-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation

In a current system, recombinant adenovirus is generated from homologous recombination between shuttle vector and provirus vector Due to the possible recombination between two proviral vectors, wild-type adenovirus may be generated from this process. Therefore, it is critical to isolate a single clone of virus from an individual plaque and examine its genomic structure

Generation and propagation of the current adenovirus vectors, which are replication deficient, depend on a unique helper cell line, designated 293, which was transformed from human embryonic kidney cells by Ad5 DNA fragments and constitutively expresses El proteins (Graham et al, 1977) Since the E3 region is dispensable from the adenovirus genome (Jones and Shenk, 1978), the current adenovirus vectors, with the help of 293 cells, carry foreign DNA in either the El, the D3 or both regions (Graham and Prevec, 1991) In nature, adenovirus can package approximately 105% of the wild-type genome (Ghosh-Choudhury et al, 1987), providing capacity for about 2 extra kB of DNA. Combined with the approximately

5.5 kB of DNA that is replaceable in the El and E3 regions, the maximum capacity of the current adenovirus vector is under 7.5 kB, or about 15% of the total length of the vector. More than 80% of the adenovirus viral genome remains in the vector backbone 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).

Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells As stated above, the preferred helper cell line is 293.

Recently, Racher et al, (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus. In one format, natural cell aggregates are grown by

inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium Following stirring at 40 φm, the cell viability is estimated with trypan blue. In another format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows. A cell inoculum, resuspended in 5 ml of medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer flask and left stationary, with occasional agitation, for 1 to 4 h The medium is then replaced with 50 ml of fresh medium and shaking initiated. For virus production, cells are allowed to grow to about 80% confluence, after which time the medium is replaced (to 25% of the final volume) and adenovirus added at an MOI of 0.05 Cultures are left stationary overnight, following which the volume is increased to 100% and shaking commenced for another 72 h.

Other than the requirement that the adenovirus vector be replication defective, or at least conditionally defective, the nature ofthe adenovirus vector is not believed to be crucial to the successful practice ofthe invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication-defective adenovirus vector for use in the present invention This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.

As stated above, the typical vector according to the present invention is replication-defective and will not have an adenovirus El region Thus, it will be most convenient to introduce the polynucleotide encoding the gene of interest at the position from which the El -coding sequences have been removed. However, the position of insertion of the construct within the adenovirus sequences is not critical to the invention. The polynucleotide encoding the gene of interest may also be inserted in lieu 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

No side effects have been reported in studies of vaccination with wild-type adenovirus (Couch et al, 1963; Top et al, 1971), demonstrating their safety and therapeutic potential as in vivo gene transfer vectors

Adenovirus vectors have been used in 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

(Rosenfeld et al, 1991; Rosenfeld et al, 1992), muscle injection (Ragot et al, 1993), peripheral intravenous injections (Herz and Gerard, 1993) and stereotactic inoculation into the brain (Le Gal La Salle et al, 1993)

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. Two long terminal repeat (LTR) sequences are present at the 5' and 3' ends of the viral genome These contain strong promoter and enhancer sequences and are also required for integration in the host cell genome (Coffin, 1990)

In order to construct a retroviral vector, a nucleic acid encoding a gene of interest is inserted into the viral genome in the place of certain viral sequences to produce a virus that is replication-defective In order to produce virions, a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983) When a recombinant plasmid containing a cDNA, together with the retroviral LTR and packaging sequences is introduced into this cell line (by calcium phosphate precipitation for example), the packaging sequence allows the RNA transcript of the recombinant plasmid to be packaged into viral particles, which are then secreted into the culture media (Nicolas and Rubenstein, 1988, Temin, 1986; Mann et al, 1983). The media containing the recombinant retroviruses is then collected, optionally concentrated, and used for gene transfer Retroviral vectors are able to infect a broad variety of cell types However, integration and stable expression require the division of host cells (Paskind et al, 1975).

A novel approach designed to allow specific targeting of retrovirus vectors was recently developed based on the chemical modification of a retrovirus by the chemical addition of lactose residues to the viral envelope. This modification could permit the specific infection of hepatocytes via sialoglycoprotein receptors

A different approach to targeting of recombinant retroviruses was designed in which biotinylated antibodies against a retroviral envelope protein and against a specific cell receptor were used. The antibodies were coupled via the biotin components by using streptavidin (Roux et al, 1989). Using antibodies against major histocompatibility complex class I and class II antigens, they demonstrated the infection of a variety of human cells that bore those surface antigens with an ecotropic virus in vitro (Roux et al, 1989)

There are certain limitations to the use of retrovirus vectors in all aspects ofthe present invention. For example, 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 However, new packaging cell lines are now available that should greatly decrease the likelihood of recombination (Markowitz et al, 1988, Hersdorffer et al, 1990)

Other viral vectors may be employed as expression constructs in the present invention Vectors derived from viruses such as vaccinia virus (Ridgeway, 1988,

Baichwal and Sugden, 1986, Coupar et al, 1988) adeno-associated virus (AAV) (Ridgeway, 1988, Baichwal and Sugden, 1986, Hermonat and Muzycska, 1984) and herpesviruses may be employed They offer several attractive features for various mammalian cells (Friedmann, 1989, Ridgeway, 1988, Baichwal and Sugden, 1986, Coupar et al, 1988, Horwich et al, 1990)

With the recent recognition of defective hepatitis B viruses, new insight was gained into the structure-function relationship of different viral sequences In vitro studies showed that the virus could retain the ability for helper-dependent packaging and reverse transcription despite the deletion of up to 80% of its genome (Horwich et al, 1990) This suggested that large portions of the genome could be replaced with foreign genetic material The hepatotropism and persistence (integration) were particularly attractive properties for liver-directed gene transfer Chang et al, recently introduced the chloramphenicol acetyltransferase (CAT) gene into duck hepatitis B virus genome in the place of the polymerase, surface, and pre- surface coding sequences It was co-transfected with wild-type virus into an avian hepatoma cell line Culture media containing high titers of the recombinant virus were used to infect primary duckling hepatocytes Stable CAT gene expression was detected for at least 24 days after transfection (Chang et al, 1991)

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

Several non-viral methods for the transfer of expression constructs into cultured mammalian cells also are contemplated by the present invention These 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,

1979) and lipofectamine-DNA complexes, cell sonication (Fechheimer et al, 1987), gene bombardment using high velocity microprojectiles (Yang et al, 1990), and receptor-mediated transfection (Wu and Wu, 1987, Wu and Wu, 1988) Some of these techniques may be successfully adapted for in vivo or ex vivo use

Once the expression construct has been delivered into the cell the nucleic acid encoding the gene of interest may be positioned and expressed at different sites. In certain embodiments, 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) In yet further embodiments, 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

In yet another embodiment of the invention, 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

In still another embodiment of the invention for transferring a naked DNA expression construct into cells may involve particle bombardment This method depends on the ability to accelerate DNA-coated microprojectiles to a high velocity allowing them to pierce cell membranes and enter cells without killing them (Klein et al, 1987) Several devices for accelerating small particles have been developed One such device relies on a high voltage discharge to generate an electrical current, which in turn provides the motive force (Yang et al, 1990) The microprojectiles used have consisted of biologically inert substances such as tungsten or gold beads

Selected organs including the liver, skin, and muscle tissue of rats and mice have been bombarded in vivo (Yang et al, 1990, Zelenin et al, 1991) This may require surgical exposure of the tissue or cells, to eliminate any intervening tissue between the gun and the target organ, i.e., ex vivo treatment Again, DNA encoding a particular gene may be delivered via this method and still be incoφorated by the present invention

In a further embodiment of the invention, the expression construct may be entrapped in a liposome Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium Multilamellar liposomes have multiple lipid layers separated by aqueous medium They form spontaneously when phospholipids are suspended in an excess of aqueous solution The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991) Also contemplated 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,

HeLa and hepatoma cells Nicolau et al, (1987) accomplished successful liposome- mediated gene transfer in rats after intravenous injection

In certain embodiments of the invention, 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) In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991) In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG- 1 In that such 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 Where a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase

Other expression constructs which can be employed to deliver a nucleic acid encoding a particular gene into cells are 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). Recently, a synthetic neoglycoprotein, which recognizes the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol et al, 1993; Perales et al, 1994) and epidermal growth factor (EGF) has also been used to deliver genes to squamous carcinoma cells (Myers, EPO 0273085)

In other embodiments, the delivery vehicle may comprise a ligand and a liposome For example, Nicolau et al, (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, incoφorated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes Thus, it is feasible that 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. For example, epidermal growth factor (EGF) 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 Also, antibodies to CD5 (CLL), CD22 (lymphoma), CD25 (T-cell leukemia) and MAA (melanoma) can similarly be used as targeting moieties

In certain embodiments, 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

C. Methods of Cloning a FR-MP Gene

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 Various embodiments may be employed to achieve this cloning

Specifically envision are the use of 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 In hybrid- arrested translation, 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

D. Diagnostic Methods

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

A. Genetic Diagnosis

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 Further, it is contemplated that 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 In one embodiment, the RNA is whole cell RNA, in another, it is poly-A RNA Normally, the nucleic acid is amplified

Depending on the format, the specific nucleic acid of interest is identified in the sample directly using amplification or with a second, known nucleic acid following amplification Next, the identified product is detected In certain applications, the detection may be performed by visual means (e.g., ethidium bromide staining of a gel)

Alternatively, 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)

Following detection, one may compare the results seen in a given patient with a statistically significant reference group of normal patients and patients that have FR- MP-related pathologies In this way, it is possible to correlate the amount or kind of FR-MP detected with various clinical states

Various types of defects are to be identified Thus, "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

A variety of different assays are contemplated in this regard, including but not limited to, fluorescent in situ hybridization (FISH), direct DNA sequencing, PFGE analysis, Southern or Northern blotting, single-stranded conformation analysis (SSCA),

RNAse protection assay, allele-specific oligonucleotide (ASO), dot blot analysis, denaturing gradient gel electrophoresis, RFLP and PCR-SSCP

(i) Primers and Probes The term 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 Typically, 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 prefeπed 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

In preferred embodiments, 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)

(ii) Template Dependent Amplification Methods

A number of template dependent processes are available to amplify the marker sequences present in a given template sample One of the best known amplification methods is the polymerase chain reaction (referred to as PCR™) which is described in detail in U.S. Patent Nos. 4,683,195, 4,683,202 and 4,800,159, and in Innis et al,

1990, each of which is incoφorated herein by reference in its entirety

Briefly, in 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 If the marker sequence is present in a sample, 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 By raising and lowering the temperature of the reaction mixture, 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

Another method for amplification is the ligase chain reaction ("LCR"), disclosed in EPO No 320 308, incoφorated herein by reference in its entirety Qbeta Replicase, described in PCT Application No. PCT/US87/00880, may also be used as still another amplification method in the present invention An isothermal amplification method, in which restriction endonucleases and ligases are used to achieve the amplification of target molecules that contain nucleotide 5'-[alpha-thio]-triphosphates in one strand of a restriction site may also be useful in the amplification of nucleic acids in the present invention, Walker et al, (1992) Strand Displacement Amplification (SDA) is another method of carrying out isothermal amplification of nucleic acids which involves multiple rounds of strand displacement and synthesis, i.e., nick translation A similar method, called Repair Chain Reaction (RCR), involves annealing several probes throughout a region targeted for amplification, followed by a repair reaction in which only two of the four bases are present. The other two bases can be added as biotinylated derivatives for easy detection A similar approach is used in SDA Target specific sequences can also be detected using a cyclic probe reaction (CPR) Still another amplification methods described in GB Application No 2 202 328, and in PCT Application No PCT/US89/01025, each of which is incoφorated herein by reference in its entirety, may be used in accordance with the present invention Other 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, incoφorated herein by reference in their entirety) Davey et al, EPO No 329 822 (incoφorated herein by reference in its entirety) disclose a nucleic acid amplification process involving cyclically synthesizing single- stranded RNA ("ssRNA"), ssDNA, and double-stranded DNA (dsDNA), which may be used in accordance with the present invention Miller et al, PCT Application WO 89/06700 (incorporated herein by reference in its entirety) disclose a nucleic acid sequence amplification scheme based on the hybridization of a promoter/primer sequence to a target single-stranded DNA ("ssDNA") followed by transcnption of many RNA copies of the sequence This scheme is not cyclic, i.e., new templates are not produced from the resultant RNA transcripts Other amplification methods include "RACE" and "one¬ sided PCR" (Frohman, M.A., In PCR PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press, N Y , 1990, Ohara et al, 1989, each herein incoφorated by reference in their entirety)

Methods based on ligation of two (or more) oligonucleotides in the presence of nucleic acid having the sequence of the resulting "di-oligonucleotide", thereby amplifying the di-oligonucleotide, may also be used in the amplification step of the present invention Wu et al, (1989), incorporated herein by reference in its entirety

(iii) 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

Briefly, 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

Subsequently, 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.

(iv) Separation Methods

It normally is desirable, at one stage or another, to separate the amplification product from the template and the excess primer for the purpose of determining whether specific amplification has occurred. In one embodiment, amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al, 1989.

Alternatively, chromatographic techniques may be employed to effect separation. There are many kinds of chromatography which may be used in the present invention: adsoφtion, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography (Freifelder, 1982).

(v) Detection Methods

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. Alternatively, if the amplification products are integrally labeled with radio- or fluorometrically-labeled nucleotides, the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Following separation of amplification products, 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. In another embodiment, 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

In one embodiment, detection is by a labeled probe The techniques involved are well known to those of skill in the art and can be found in many standard books on molecular protocols See Sambrook et al, 1989 For example, chromophore or radiolabel probes or primers identify the target during or following amplification

One example of the foregoing is described in U S Patent No 5,279,721, incoφorated by reference herein, which discloses an apparatus and method for the automated electrophoresis and transfer of nucleic acids The apparatus permits electrophoresis and blotting without external manipulation of the gel and is ideally suited to carrying out methods according to the present invention

In addition, the amplification products described above may be subjected to sequence analysis to identify specific kinds of variations using standard sequence analysis techniques Within certain methods, exhaustive analysis of genes is carried out by sequence analysis using primer sets designed for optimal sequencing (Pignon et al, 1994) The present invention provides methods by which any or all of these types of analyses may be used Using the sequences disclosed herein, oligonucleotide primers may be designed to permit the amplification of sequences throughout an FR- MP gene that may then be analyzed by direct sequencing

(vi) Kit Components All the essential materials and reagents required for detecting and sequencing

FR-MP and variants thereof may be assembled together in a kit This generally will comprise preselected primers and probes Also included may be enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, Sequenase™ etc ), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification Such 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. Immunodiagnosis

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

The use of antibodies of the present invention, in an ELISA assay is contemplated For example, 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 After washing to remove incompletely adsorbed material, it is desirable to bind or coat the assay plate wells with 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 This allows for blocking of non-specific adsorption sites on the immobilizing surface and thus reduces the background caused by non-specific binding of antigen onto the 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.

Following formation of specific immunocomplexes between the test sample and the bound antibody, and subsequent washing, 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 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 ^® or borate buffer.

To provide a detecting means, the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate. Thus, for example, 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 ^® ).

After incubation with the second enzyme-tagged antibody, and subsequent to washing to remove unbound material, the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol puφle 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 In conjunction with 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

C. Methods for FR-MP Activity In addition to the genetic and immunodiagnostic procedures outlined above, one may assay for FR-MP proteolytic activity. The procedures for measuring FR-MP activity are outlined elsewhere in this document

E. Therapeutic Methods Metalloprotease compositions of the invention may be used in therapeutic methods or studies For example, 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) In addition, generally speaking, the metalloproteases may be used for puφoses related to those for which a range of antifolates are used and/or to study the action of such antifolates. Because such folate receptor-directed metalloproteases are known to occur in cervical cancer cells, 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.

When enough FR are expressed, these proteins mediate the uptake of

5-methyl-tetrahydrofolate and antifolates (such as methotrexate) with comparable rates to cells expressing only the reduced-folate carrier (Spinella et al, 1995). Thus, 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

Further, the elucidation of a new pathway between FR expression and TK appears to have major biological implications for the use of conventional antifolates that rely on reduction of thymidylate synthase, without the recruitment of salvage pathways For example, the FR-overexpressing cells become resistant to MTX and 5-FU but are more sensitive to AZT These data are convincing that this increase in TK is of major biological relevance to cancer chemotherapy involving antifolates In any event, the overexpressed FR allows the use of these proteins as Trojan horses to internalize folate-tethered liposomes bearing nucleic acids encoding transgenes, anti¬ sense oligonucleotides or cytotoxic agents such as doxorubicin, cisplatin, taxol and other chemotherapeutics well know to those of skill in the art (see "Remington's Pharmaceutical Sciences" 15th Edition)

Thus, 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 In addition, an 18-mer antisense molecule has been shown to effect synthesis of FR-α also has proved to be effective at inhibiting FR expression

F. Therapeutic Compositions and Routes 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

One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells Buffers also will be

employed when recombinant cells are introduced into a patient Aqueous 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 Such compositions also are referred to as inocula The phrase "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents and the like The use of such media and agents for pharmaceutically active substances is well know in the art

Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated Supplementary active ingredients also can be incoφorated into the compositions

The active 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

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 syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The 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. In many cases, it will be preferable to include 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 absoφtion, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incoφorating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incoφorating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the 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.

As used herein, "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.

For oral administration the polypeptides of the present invention may be incoφorated 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) Alternatively, the active ingredient may be incoφorated 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.

The 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.

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.

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 These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure For example, one dosage could be dissolved in 1 ml of isotonic 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 The person responsible for administration will, in any event, determine the appropriate dose for the individual subject Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards

In a particular embodiment, 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 has been exploited with so-called "Trojan horses" to mediate the cellular uptake of folate conjugates for therapeutic and diagnostic use (Leamon and Low,

1991, Leamon and Low, 1993, Leamon et al, 1993, Leamon and Low, 1994, Lee and Low, 1994; Lee and Low, 1995) By conjugation of liposomes to folate using polyethylene glycol spacers, several hundred folate-tethers were built into each liposome This led to multiple simultaneous attachments to cell surface FRs with a significant increase in binding affinity to FR and selective targeting to high

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) Thus, 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. 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 cationic lipid-nucleic acid complexes, such as lipofectamine-nucleic acid complexes

In certain embodiments of the invention, 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) In other embodiments, the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991) In yet further embodiments, the liposome may be complexed or employed in conjunction with both HVJ and HMG-1 In that such 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 Where 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 ("DMPC") can be obtained from Sigma Chemical Co., dicetyl phosphate ("DCP") is obtained from K & K Laboratories (Plainview, NY), cholesterol ("Choi") is obtained from Calbiochem-Behring, dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti

Polar Lipids, Inc (Birmingham, Ala ) Stock solutions of lipids in chloroform, chloroform/methanol or t-butanol can be stored at about -20°C Preferably, chloroform is used as the only solvent since it is more readily evaporated than methanol

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. In aqueous suspension, 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 For example, when aqueous phases are present both within and without the liposome, 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 In one preferred embodiment, liposomes are prepared by mixing liposomal lipids, in a solvent in 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

In the alternative, liposomes can be prepared in accordance with other known laboratory procedures the method of Bangham et al (1965), the contents of which are incoφorated 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 incoφorated herein by reference, the method of Deamer and Uster (1983), the contents of which are incoφorated 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

In a preferred embodiment, 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)

G. Screening for Active Compounds

A . Inhibitors of FR-MP A ctivity

In one embodiment of the present invention, 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

First, one may 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 Second, one may perform binding studies between a candidate substance and FR-MP These assays may examine the mobility shift, in gel electrophoresis, of either FR-MP or the candidate Third, where the candidate inhibitor is predicted to reduce the amount of FR-MP expressed, or to alter the processing of FR-MP, one may look directly at FR-MP in its various forms Typically, 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

In some cases, the candidate inhibitor substance may be contacted with the cell directly In other situations, depending on the nature and putative mechanism of action, the candidate inhibitor substance may be reformulated to provide improved uptake For example, where antisense oligonucleotides are provided, these may advantageously be formulated in liposomes or as virally-encapsulated expression

vehicles. Where polypeptides are to be tested, it may be advantageous to provide expression vectors encoding these molecules rather than the polypeptides themselves. Essentially, the most reasonable mechanism for delivering an effective amount of the candidate inhibitor substance to the proper intracellular site will be chosen "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.

Once the candidate inhibitor substance has been provided to a cell that expresses FR-MP, the evaluation of the effects of the inhibitor may be undertaken. Depending on the type of assay used, it may be possible to automate this process and test hundreds of candidates at the same time. For example, 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.

B. Inducers of FR-MP Activity

In another embodiment of the screening assay, one will test candidate substances for the ability to induce or enhance FR-MP activity. The formats are essentially as set forth above in screening for candidate inhibitors.

H. Antibodies to FR-MP and Uses Therefor

In another aspect, 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).

Briefly, 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. A wide range of animal species can be used for the

production of antisera Typically 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.

It is proposed that 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.

In general, 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. The operation of all such immunological techniques will be known to those of skill in the art in light ofthe present disclosure.

Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988, incorporated herein by reference) More specific examples of monoclonal antibody preparation are give in the examples below

As is well known in the art, a given composition may vary in its immunogenicity It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, /w-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine

As also is well known in the art, the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant

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

(subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal) The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved When a desired level of immunogenicity is obtained, the immunized animal can be bled and the 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, incoφorated herein by reference. Typically, 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. 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.

Following immunization, somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol. These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible. Often, a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe. Typically, a spleen from an immunized mouse contains approximately 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). For example, where the immunized animal is a mouse, one may use P3-X63/Ag8, 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

2: 1 ratio, though the ratio may vary from about 20: 1 to about 1 : 1, respectively, in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes Fusion methods using Sendai virus have been described (Kohler and Milstein, 1975; 1976), and those using polyethylene glycol (PEG), such as 37% (v/v) PEG, by Gefter et al, (1977). The use of electrically induced fusion methods is also appropriate (Goding, 1986).

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. Where aminopterin or methotrexate is used, the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium). Where azaserine is used, the media is supplemented with hypoxanthine.

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. The B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.

This culturing provides a population of hybridomas from which specific hybridomas are selected. Typically, selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supernatants (after about two to three weeks) for the desired reactivity. The assay should be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunobinding assays, and the like.

The selected hybridomas would then be serially diluted and cloned into individual antibody-producing cell lines, which clones can then be propagated indefinitely to provide mAbs. The cell lines may be exploited for mAb production in two basic ways. A sample of the hybridoma can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. 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.

I. Examples The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skilled the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor

to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should , in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept ofthe invention as defined by the appended claims.

EXAMPLE 1: Purification of and Assay for FR-MP Activity

Abbreviations: The following abbreviations are used herein: FR, folate receptor; PFR, placental folate receptor; GPI, glycosyl-phosphatidylinositol; HPLC,

High performance liquid chromatography; PteGlu, Pteroylglutamate (folic acid); Con

A, Concanavalin A; TFA, trifluoroacetic acid; FACS, Fluorescence activated cell sorting.

Assay for Placental Metalloenzyme: The assay which was carried out in

Beckman 96-well Flat Bottom Immunoassay Plates, relied on the ability of solubilized placental hydrophobic metalloenzyme to cleave mature ¹²⁵ I-hydrophobic PFR to hydrophilic forms. Both enzyme and substrate were retained in the micellar phase at the cloud point of Triton X-l 14 (Verma et al, 1992), while the hydrophilic PFR was recovered in the aqueous phase. Briefly, 350 fmol of ¹²⁵ I-hydrophobic PFR (-2 x IO ⁴ cpm in 20 μl contained -12.25 ng) was added to 100 μl of metalloenzyme in the aAence or presence of 60 mM EDTA or 60 mM EGTA in 10 mM potassium phosphate, pH 7.5, and 20 mM MgCl ₂ , in a final reaction volume of 250 μl. Each assay was routinely determined in duplicate and judged valid only when there was <5% variation from the mean. After incubation at 37°C overnight, 50 μl of 2% pre- condensed Triton X-l 14 was added, and the mixture was incubated at 4°C for 15 min to disperse micelles (Bordier, 1981) After re-incubation at 37°C for 30 min, the

regenerated micelles were sequestered in a detergent rich micellar phase of < 50 μl (Verma et al, 1992) by centrifugation of the mixture at 2750 φm for 15 min in a microplate carrier designed for a Beckman GH 3 7 rotor in a Beckman GPR table-top centrifuge Following centrifugation, 130 μl of the aqueous phase from each well which contained ¹²⁵ I-hydrophilic PFR was counted for radioactivity, and values were normalized for a total volume of 250 μl One unit of metalloenzyme activity was defined as the amount required to convert 2 fmol of ¹²⁵ I-hydrophobic PFR to hydrophilic forms in 1 min at 37°C

Purification ofthe Placental Hydrophobic PFR-directed Metalloprotease:

1 Preparation of Crude Solubilized Metalloenzyme: A full-term normal placenta following uncomplicated vaginal delivery was homogenized within 1 h with 2 volumes of 14 mM potassium phosphate, pH 7 5, at 4°C and centrifuged (30,000g for 30 min) (Antony et al, 1981) The pellet was washed with another cycle of homogenization and centrifugation before solubilization with 10 mM potassium phosphate, pH 7.5, containing 1 5% Triton X-l 14 and 20 mM MgCl ₂ for 16 h at 4°C (Antony et al, 1981) After centrifugation (30,000g for 30 min), the metalloenzyme- rich supernatant was collected

2 Temperature-Induced Phase-Separation in Triton X-l 14 Triton X-

114 was then added to achieve a final concentration of 2% and temperature-induced phase-separation at 37°C was used to generate a detergent-rich micellar phase and an aqueous supernatant (Bordier, 1981) To capture residual metalloenzyme from the supernatant, the cycle of addition of Triton X-l 14 followed by temperature-induced phase-separation was repeated, and all detergent-rich phases were combined

3 Affinity Chromatography Using Concanavalm A (Con A) - Sepharose:

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 3 volumes of 0 5 M of a-methyl D-mannoside in Con A-binding buffer, and the eluate was collected in its entirety

These steps were repeated 5 times with storage of each batch-eluate at -80°C until completion of this purification step

4 Ultrafiltration The pooled Con A-Sepharose eluates were then concentrated by ultrafiltration using a Millipore Minitan System at 4°C and Centriprep

Concentrators (30 kD cut-off filters)

5 Reverse-Phase HPLC: The Con A-Sepharose eluate (1 mg) was applied to a 1x25 cm reverse-phase C column (Vydac, Hesperia, CA) connected to a Waters HPLC system (Milford, MA) After washing with equilibration buffer consisting of 80% buffer A (0 1% trifluoroacetic acid (TFA) in water) at a flow rate of 5 ml/min for 5 min, the column was eluted using a gentle "convex-up" (program #4) gradient increase of buffer B (75% isopropanol/20% acetonitrile/5% water containing 0.1% TFA, final pH 3 2) from 20% to 60% over 25 mins The fraction corresponding to a peak of protein eluted by 51% buffer B (3 ml total) was collected in 100 μl of 1 M Tris-HCl, pH 8 0, containing 100 mM MgCl ₂ , and aliquots were assayed for activity and protein, and analyzed by SDS-PAGE (Verma and Antony, 1991)

Characterization ofthe Metalloenzyme: Gel Filtration Analysis of Metalloenzyme Activity: ¹²⁵ I-hydrophobic PFR,

30,000 cpm, was incubated with Con A-Sepharose eluate (40 μg) and HPLC-purified metalloenzyme (6 to 20 μg) in a final volume of 200 μl of buffer consisting of 10 mM potassium phosphate, pH 7.5, 20 mM MgCl ₂ , 0 1% Triton X-l 14 in the presence or absence of 100 mM EDTA, and conversion to hydrophilic PFR was analyzed by Sephacryl S-200 gel filtration in Triton X- 100 (Henderson et al. , 1995)

Radiolabeling Studies Purified metalloenzyme (20 μg) and hydrophobic PFR (10 μg) (Verman et al 1992) were iodinated (Verma and Antony, 1991), and free [ ¹²⁵ I]NaI was removed by passage of the reaction mixture over a NucTrap push column (Stratagene, La Jolla, CA) Assuming 100% recovery, the specific activity of ¹²⁵ I-metalloprotease and ¹²⁵ I-hydrophobic PFR was 0 13 μCi/μg and 1 16 μCi/μg, respectively Hydrophobic PFR (Verma et al, 1992) was also covalently-labeled within its ligand-binding site with the N-hydroxysuccinimide ester of [ ³ H]PteGlu (Henderson and Zevely, 1984) Based on a 1 1 molar stoichiometry and assuming 100% recovery, the specific activity of [ ³ H]PteGlu-labeled hydrophobic PFR was 2 87 Ci/mmol

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 unincoφorated [ 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

Amino Acid Analysis: Analysis of purified metalloenzyme was carried out after vapor hydrolysis and pre-column phenylisothiocyanate derivatization (Moore and Stein, 1963) Briefly, 10 μg was hydrolyzed at 165°C in 600 μl of 6N HCl

Phenylthiocarbamyl amino acids were analyzed on an Applied Biosystems 130A Separation System using a 2 1 x 220 mm Brownlee Cι ₈ column (Foster City, CA) Phenylthiocarbamyl amino acid peaks were calibrated with the Pierce Amino acid Standard H

Endoproteolytic Cleavage of In Vitro Translated Nascent PFR by Metalloprotease The 1 1 kB PFR cDNA generously provided by Dr P C Elwood

(Medicine Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD) (Elwood, 1989) was cloned into the EcoRI site of the transcription facilitating vector, pSPT18 (Pharmacia, Piscataway, NJ) Recombinant plasmid DNA was then expanded, purified, and PFR cDNA was recovered by restriction with Bglll (Sambrook et al, 1989). 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 μg of PFR mRNA was translated in vitro in the presence of 8 μCi of [ ³ H]leucine, and 40 μl 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 μg of metalloenzyme (Con A-Sepharose eluate) in the absence or presence of 60 mM EDTA in a final volume of 220 μl 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 (0.1% TFA in water) and 5% buffer C (0 1% TFA in water, 95% acetonitrile) After basal conditions of perfusion were continued for 5 min, a linear gradient (from 5% to 100% buffer C over 55 min) was established Fractions collected at a flow rate of 5 ml min/fraction were counted for radioactivity A second aliquot was analyzed by Sephacryl S-200 gel filtration in Triton X-l 00 (Verma et al 1992) A third aliquot (10,000 cpm) was reacted with either 20 μl of nonimmune serum or anti-hydrophobic PFR antiserum, and the extent of binding of antigen-antibody complexes to Protein A was determined (Antony et al, 1987, Antony et al, 1987)

Preparation of Anti-Metalloprotease Antiserum A 12 week-old female New Zealand white rabbit was immunized with 250 μg of purified metalloprotease (Antony et al, 1982) and antiserum was prepared (Antony et al, 1981)

Fluorescence Activated Cell Sorting (FACS) Analysis Human bone marrow cells were aspirated from the posterior iliac crest of a hematologically normal volunteer according to guidelines established by the Institutional Review Board of Indiana

University School of Medicine. Freshly-harvested low-density mononuclear human bone marrow cells (1 x IO ⁵ ) (Antony et al, 1991), and human cervical carcinoma

[HeLa-IUi cells (Sun et al, 1995)], (5 x IO ⁶ ), were first labeled with either 100 μl of anti-metalloprotease antiserum or anti-hydrophobic PFR antiserum (at 1:5 dilution in nonimmune serum), or 100 μl of nonimmune serum followed by labeling with second antibody (goat anti-rabbit IgG labeled with fluorescein isothiocyanate), cells were finally analyzed by FACS (Antony et al. , 1991 )

Assay for the Solubilized Placental Metalloenzyme: In preliminary studies, the specific conversion of GPI-anchored ^I25 I-hydrophobic PFR (which sequesters in the detergent-rich, Triton X-l 14 micellar phase) to ¹²⁵ I-hydrophilic PFR (released into the aqueous phase) using the temperature-induced phase-separation assay was verified by the gel filtration assay in Triton X-l 00 (Antony et al, 1989). In addition, when the ligand-binding site of hydrophobic PFR was covalently-labeled with the N- hydroxysuccinimide ester of [ ³ H]PteGlu, metalloenzyme-mediated cleavage of this substrate also led to release ofthe [ ³ H]PteGlu-bound hydrophilic PFR into the aqueous phase (Antony et al, 1989). Thus, this phase-separation assay appropriately reflected the conversion of hydrophobic PFR to hydrophilic forms consistent with the activity of the putative FR-directed metalloprotease.

Identification of Distinct EDTA-sensitive and EDTA-insensitive PFR- directed Enzymes: Dose-response studies using a fixed amount of [ ³ H]PteGlu-labeled hydrophobic FR incubated with increasing concentrations of crude enzyme revealed that -50% conversion to hydrophilic PFR was achieved with 62.4 μg of crude enzyme, and 90% conversion achieved with 400 μg (FIG. IA) While a major portion of the crude metalloenzyme at low concentrations (<40 μg protein) was EDTA-sensitive, at higher concentrations (>200 μg protein), nearly equivalent amounts of conversion was accounted for by an EDTA-insensitive activity, suggesting the presence of another enzyme which cleaved hydrophobic PFR. Alternatively, this could be a function of reversal of the chelating effect of EDTA (present in fixed concentrations) by the addition of increasing amounts of Mg ²⁺ (extant in the crude enzyme preparation) to the reaction mixture; these possibilities were further investigated From 5 different crude metalloenzyme preparations, on average, 74% of substrate was converted under basal conditions of which 36% was EDTA-insensitive When this preparation was phase-

separated at the cloud point of Triton X-l 14 and the micellar and aqueous phases ere individually assayed, the latter was enriched for the EDTA-insensitive enzyme, i.e., 48% of total conversion was not inhibited by EDTA More importantly however, the micellar phase was significantly enriched for the EDTA-sensitive enzyme since only 5% of converting activity was EDTA-insensitive While these data confirmed the presence of two distinct placental enzymes which converted hydrophobic PFR to hydrophilic forms, they also supported the conclusion that the EDTA-sensitive enzyme (metalloenzyme) was hydrophobic while the EDTA-insensitive enzyme was hydrophilic

The optimal duration of incubation at 37°C was determined for two levels of crude metalloenzyme (FIG IB) The basal conversion recorded for the higher level of metalloenzyme likely occurred during the incubation at 37°C to effect temperature- induced phase-separation Following incubation for 2 h, there was 60% and 90% conversion of hydrophobic PFR to hydrophilic PFR at 400 μg and 2000 μg of metalloenzyme, respectively. Thus, 2 h was optimum, with no additional gain even after incubation for 16-24 h, consistent with earlier data (Antony et al, 1989)

In preliminary studies, an activity which bound to and was eluted from Con A- Sepharose was noted to be almost entirely (>95%) sensitive to EDTA. SDS-PAGE of this preparation revealed a major protein which migrated at 63,000 M,. When the Con- A-Sepharose eluate was subjected to HPLC gel filtration in Triton X-l 14, and each eluted fraction was individually tested for metalloenzyme activity, there was a major protein peak with activity which also migrated on SDS-PAGE at 63 kD Thus, this species was the likely metalloenzyme

Isolation of the Hydrophobic Metalloenzyme: Table 4 reveals the purification scheme used to isolate the metalloenzyme 267-fold with a final yield of 16% from human placenta The isolated metalloenzyme eluted as a single protein peak at a concentration of 51 % buffer B on reverse-phase HPLC (FIG. 2 A) SDS-PAGE of this preparation revealed a single protein band of 63,000 M _r , which reduced to 58,000 M _r After deglycosylation with recombinant glycopeptidase F (FIG 2B) In addition,

temperature-induced phase-separation assay of this sample revealed a dose-dependent release of ¹²⁵ I-hydrophobic PFR into the aqueous phase (FIG. 2C). Furthermore, the ^I25 I-hydrophobic PFR with HPLC-purified metalloprotease and temperature-induced phase-separation) exhibited an elution profile consistent with that of hydrophilic PFR on gel filtration analysis in Triton X-l 00 (Henderson et al, 1995) Together, these data indicated that the HPLC-purified metalloprotease was functionally active.

When 20 μg of this preparation was iodinated, a single iodinated peak was identified on SW300 HPLC gel filtration which also migrated as a 63 kD species on SDS-PAGE. Interestingly, although both crude and Con A-Sepharose-eluted metalloenzyme exhibited ^{1 5} I-hydrophobic PFR-directed activity on Sephacryl S-200 gel filtration in Triton X-l 00 (Henderson et al, 1995), similar activity could not be clearly documented using the isolated 63-kDa metalloenzyme. Instead, the products of the reaction mixture consistently yielded a single ¹²⁵ I peak which eluted at the same position as ¹²⁵ I-hydrophobic PFR Since this was not due to the lack of activity of the

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.

Table 4 Summary of Purification of Metalloprotease from Human Placenta ⁸

Step Volume Protein Total Specific Recovery Purification

(ml) (mg) units activity (%) (fold)

(units/mg

)

1 Solubilized 815 3261 8153 2 5 100 pellet

2 Triton X-l 14 250 1400 4375 3 1 54 1 2 phase-separation

3 Concanavalin 250 200 3125 15 6 38 A-Sepharose eluate

4 Ultrafiltration 50 56

5 HPLC peak 2 1333 667 0 16 267 fraction

"The data represents the purification profile starting with one placenta ^b Percent recovery = total unitS _f rac _tιon /8153

Characterization of the Metalloenzyme The partially-purified Con A- Sepharose eluate was used to characterize the enzyme's activity under controlled conditions

1 Rate of Conversion FIG 3 A shows the conversion of ¹²⁵ I-hydrophobic

PFR to hydrophilic PFR by a fixed amount of metalloenzyme as a function of increasing incubation times Although relatively less active when compared to crude enzyme (FIG IB), the rate of conversion was linear as a function of time, by 8 h, 91% conversion to hydrophilic PFR was achieved

2 Dose-Response Studies Using a fixed amount of ¹²⁵ I-hydrophobιc

PFR, conversion as a function of does of metalloenzyme was determined Thus, 50%

conversion was achieved by 10 μg of metalloenzyme and maximal conversion with 25 μg (FIG 3B)

3 Determination of the pH Optimum Conversion of ¹²⁵ I-hydrophobic PFR at pH 4, 6, 7 5, 8 and 10 was 56%, 74%, 100%, 80%, and 68%, respectively

(FIG 3C) When EDTA sensitivity was also tested at these pH, there was significant inhibition of conversion at pH 7 5 Thus, the EDTA-sensitive metalloenzyme was maximally active at physiological pH

4 Effect of Inhibitors and Reactivation by Divalent Cations The metalloenzyme was inhibited by 1,10-phenanthroline in a dose-dependent manner (FIG 3D) In addition, after incubation with EDTA which inhibited enzyme activity -95%, increasing concentrations of Mg ⁺ , Ca ²⁺ , Mn ²⁺ and Zn ²⁺ overcame the inhibition in a dose-dependent manner (FIG 3E) Although inhibition was also achieved with EGTA, and the pattern of re-activation with Ca ²⁺ or Mn ²⁺ were similar to that using

EDTA, there was no major recovery despite addition of 400 mM of Mg ²⁺ (FIG 3F) In addition, the data with Zn ²⁺ indicated that although progressively increasing concentrations up to 4 mM led to reactivation ofthe enzyme at a lower dose compared to enzyme reactivation in the presence of EDTA, concentrations >4 mM led to progressive inhibition of enzyme activity

5 Amino Acid Analysis: 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

6 Evidence that the Purified Metalloenzyme is a Protease: Since neither gel filtration nor temperature-induced phase-separation assays distinguished EDTA- sensitive protease from phospholipase activity, an important question was whether the hydrophobic 63 kD human placental metalloenzyme was a protease or GPI-specific

phospholipase Prompted by an earlier report (Elwood et al, 1991), we hypothesized that if PFR was labeled with [ ³ H]leucine during in vitro translation of PFR mRNA in the absence of microsomes, all 22 leucine residues in the nascent PFR polypeptide (Elwood, 1989) could be replaced by radiolabel, and the full length [ ³ H]leucine-labeled nascent PFR polypeptide (deduced M _r of 29,817) would also contain no post¬ translational modifications, including GPI anchor addition (Antony and Miller, 1994, Udenfriend and Kodukula, 1995) Therefore, we reasoned that EDTA-sensitive cleavage of this nascent polypeptide (especially a loss of the C-terminal [ ³ H]leucine- rich fragment) would lead to a net reduction in counts from the major fragment of [ ³ H]leucine-labeled PFR polypeptide and an alteration in its hydrophobicity leading to an altered reverse-phase HPLC elution profile

Following in vitro translation of full length 1 kB nascent PFR mRNA, -5% to 10% of total ] ³ H]leucine integrated into nascent PFR polypeptide The M _r of this species was -30,000 on SDS-PAGE, and it eluted at apparent M _r of -80,000 on

Sephacryl S-200 gel filtration analysis in Triton X-l 00, findings distinctly different from mature GPI-anchored PFR [which migrated on SDS-PAGE at 44,000 M _r and eluted at apparent 160,000 M _r , respectively] (Verma et al, 1992) Furthermore, although -1 μl of anti-PFR antiserum immunoprecipitated >90% of mature [ ¹²⁵ I]PteGlu (histamine derivative)-labeled FR (Verma et al, 1992, Antony et al,

1987, Antony et al, 1987), at least 20 μl of this antiserum was required to specifically immunoprecipitate [ ³ H]leucine-labeled nascent PFR polypeptide Thus, not unexpectedly, [ ³ H]leucine-labeled nascent FR polypeptide had distinct differences on SDS-PAGE, gel filtration profile in Triton X-l 00, and antigenic determinants when compared to mature PFR.

The reverse-phase HPLC elution profile of in vitro translated and nascent PFR polypeptide generated in the absence of microsomes indicated a single shaφ peak of radioactivity which eluted at 5 min When this species was incubated with 50 μg of metalloenzyme, there was a clear-cut shift of the peak to a retention time of 10 min. This conversion was EDTA-sensitive since the reaction was completely inhibited in its presence In addition, the total counts in the major peak eluting at 10 min (i.e., the

product) constituted 73% of the total counts in the EDTA-inhibited control (precursor), confirming that the alteration in profile was a function of loss of net radioactivity from the nascent PFR polypeptide. We could not locate the putative cleaved [ ³ H]leucine-labeled C-terminal fragment released from the nascent polypeptide Since the in vitro translation mixture itself contained several crude proteins some of which could be nonspecific proteases, it is possible that this smaller hydrophobic species was degraded shortly after it was released from the major body of the nascent PFR polypeptide by the metalloprotease. Therefore, since it cleaved and shifted the elution profile of [ H]leucine-labeled nascent PFR polypeptide and reduced net cpm in the major fragment, these data supported the conclusion that the metalloenzyme was a protease, and hence a metalloprotease.

Studies with Anti-Metalloprotease Antiserum: An immunoprecipitation curve involving the reaction of ¹² T-metalloprotease with increasing concentrations of anti- metalloprotease antiserum revealed a dose-dependent immunoprecipitation of ¹²⁵ I]- metalloprotease with antiserum under conditions where nonimmune serum led to (nonspecific) precipitation of <10% of the radioactivity. Thus, this antiserum contained antibodies which recognized epitopes on the isolated placental metalloprotease. HeLa-IUj cells contain GPI-anchored FR on plasma membranes (Sun et al, 1995), and an activity which was consistent with a membrane-associated metalloenzyme. Therefore, we determined whether the putative metalloenzyme in these cells would be recognized by anti-metalloprotease antiserum using FACS/fluorescence microscopy. As shown in FIG. 4, when compared to nonimmune serum, two levels of anti-metalloprotease antiserum led to a progressive shift of the fluorescence to the right by more than 10-fold and 100-fold, respectively When quantitated using mean channel fluorescence intensity, nonimmune serum gave a value o ^f 9, whereas 1: 10 dilution of anti-metalloprotease antiserum gave a value of 341 and undiluted antiserum gave a value of 1982. 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 However, anti-PFR antiserum and anti-metalloprotease antiserum gave values of 81 and 30 units, respectively Thus, 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

Since 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

EXAMPLE 2: Diagnostic Applications

The existence of a FR-MP has been demonstrated in nasopharyngeal KB cells

(Elwood et al, 1991) and HeLa-IUi cells (Yang et al, 1996) The role this protease plays in regulation of FR expression on the cell surface is not clearly elucidated, however Now the placental FR-MP has been isolated and specific antiserum has been raised, thereby permitting diagnostic evaluation of FR-MP status in human cells

It is believed that excessive FR-MP activity will release FR, and therefore be one more mechanism for down-regulation of FR This could, therefore, limit the amount of FR uptake into cells at physiological extracellular folate concentrations

Conversely, loss of FR-MP activity may lead to upregulation of FR, and therefore increase uptake of folates FR that mediate physiological cellular folate uptake are

inversely regulated by the extracellular folate concentrations Although FR up-regulation in malignant cells has been accompanied by increased FR mRNA expression, it was found that cervical carcinoma (HeLa-IUi) cells with > 10-fold FR up-regulation at low-extracellular folate concentration (designated HeLa-IUi-LF cells) expressed reduced levels FR mRNA This prompted investigations to define the underlying mechanism(s) for alteration of FR metabolism under various extracellular folate concentration When compared with high-extracellular folate concentration-adapted cells (HeLa-IUi-HF cells), HeLa-IUi-LF cells exhibited reduced transcription of FR genes, but this was partially offset by increased FR mRNA stability by -30% Quantitation of biosynthetically-labeled [ ³⁵ S]FR identified that FR synthetic rates increased 6-fold, whereas FR degradation rates were unchanged

When folate starvation was induced by propagating HeLa-IUi-HF cells in low-extracellular folate concentration, a reduction of intracellular folate concentrations to levels found in HeLa-IUi-HF cells, and a reduction of FR mRNA was observed in 2 wks However, [ ³⁵ S]SR increased only by about 8 weeks In contrast, abrupt exposure of HeLa-IUi-LF cells to high-extracellular folate concentration (various folates) for 24 h led to an -80-90% reduction in FR synthetic rates, thereby predicting eventual down-regulation of FR, however, this was accompanied by a 1 1-1 7-fold prolongation in the half-life of FR Thus, in HeLa-IUi cells, regulation of FR expression by the extracellular folate concentration is primarily controlled at the translational level through alterations in FR synthetic rates In addition, some parameters of FR metabolism were paradoxically opposite the dominant regulatory change, findings that may reflect underlying homeostatic signals that serve to limit the extent of FR regulation

Table 5

Summary of Changes in Major Parameters of FR Metabolism in HeLa-IUi Cells

Under Basal Conditions in High Folate Media (HeLa-IUi-HF) and Following

Stable Adaptation to Low-Extracellular Folate Concentration (HeLa-IUj-LF)

Parameter HeLa-IUi-HF HeLa-IUi-LF

Doubling time (h) 21.9 31.4

Nuclear: cytoplasmic ratio ³ basal unchanged

Cell surface FR (receptors/cell) 7.9 x IO ⁴ 1.5 x IO ⁶

Cell surface molecules/μm ² 78 600

Total folate-binding capacity 20 288

(pmol/mg)

Dissociation constant (nM) 0.52 0.22

FR mRNA' basal decreased by -35%

Transcription rate ³ basal decreased by -50%

FR mRNA stability" basal increased by -30%

Rate of FR synthesis (pmol*/mg/h) 0.36 2.17

Rate of FR degradation (t' ^Λ ) 24-h 26-h

Hydrophilic FR released into the 0.013 0.035 media (pmol*/mg) "Data is compared to basal conditions of culture (of HeLa-IUi-HF cells). *pmol of cysteine

These data demonstrate, for the first time, that post-transcriptional mechanisms (at the translational level) are important in the regulation of FR in these cells. This work also suggests that acute down regulation of FR is yet another cause for the development of antifolate resistance. Another important aspect to this work is that all methods are in place to analyze the major parameters of FR metabolism including quantitative analysis of FR-MP activity on FR (i.e., release into the growth media). So when FR-MP is inactivated by overexpression of antisense FR-MP cDNA, the parameters of FR metabolism leading to a relative up-regulation will be able to be quantitated and compared to mock transduced controls.

EXAMPLE 3: FR Expression in Cancer - TK, AZT and MTX Considerations

Contrary to published data, it has been determined that increased FR expression in cervical carcinoma cells leads to an increase in TK and consequent methotrexate resistance This new interrelationship in cervical carcinoma cells is highly significant to chemotherapy and is discussed below in more detail than other recently published material It also points out a possible role for FR-MP in malignancy and multi-drug resistance

Cervical carcinoma is an acquired immunodeficiency syndrome (AIDS)-defining illness The expression of 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) Although sense cells proliferated slower than antisense or untransduced cells, [methyl- ³ H]thymidine incoφoration into

DNA was significantly increased; therefore the basis for this apparently paradoxical finding was investigated When compared to antisense and untransduced cells, sense cells had a significantly greater [methyl- ³ H]thymidine incoφoration per cell number, per mg protein, and per μg DNA

The activity of thymidine kinase (TK) was subsequently directly correlated with FR expression in single cell-derived clones of transduced cells This elevated TK activity was not a result of recruitment of the salvage pathway based on significantly increased deoxythymidine triphosphate pools, normal thymidylate synthase activity, persistence of increased thymidine uptake despite provision of excess

5,10-methylene-tetrahydrofolate precursor, and documentation of more folates in sense cells

The increase in 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. The direct correlation of 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.

Surprisingly, despite slower proliferation (Sun et al, 1995), sense cells had greater [methyl - ³ H]thymidine incorporation into DNA compared to antisense and untransduced cells. Since a difference in cell number was not identified by 5 days in culture (Matsue et al, 1992), the plateau and progressive reduction in [methyl- ³ H]thymidine incoφoration in sense cells beyond day 3 in culture was not due to nutrient depletion or contact inhibition. When determined as a function of either cell number, mg protein or μg DNA (Table 6), a consistent and correlated increase in incoφoration into DNA was noted for sense cells ( 0.001) when compared to antisense and untransduced cells. Furthermore, sense cells had significantly higher TK activity when compared to both untransduced and antisense cells (Table 6). There was no statistically significant difference in these values between antisense and untransduced cells.

Table 6

Incorporation of [methyl- ³ H]Thymidine into DNA and thymidine kinase (TK) activity of untransduced, sense, and antisense cells ³

Cell lines Incorporation into DNA (cpm x 10 ) Thymidine

Kinase Activity per 10 ⁶ cells per mg per μg DNA nmol/h/mg protein protein

1 Untransduced 3 7 ± 0 5 19 9 ± 4 1 0 8 + 0 1 122 9 ± 2 5 cells

2 Sense cells 11 0 ± 1.2** 40 3 ± 3 9** 2.2 ± 0 3** 149 O ± 14 6*

3 Antisense 5 7 ± 0 2 27 9 ± 4 4 1 1 ± 0.2 1115 ± 5 9 cells

³ Mean and S D from studies carried out in triplicate

* Significantly different compared to untransduced cells (p<0.05)

** Significantly different compared to untransduced cells (p<0 001). Data using non- clonal mixtures of transduced or untransduced cells

When single cell clones of sense and antisense cell lines containing varying expression of FR (Sun et al. 1995) were analyzed for TK activity, there was a significant correlation [r = - 62 (n=19), p<0 005 (FIG 5) indicating a direct relationship between transduced FR genes (gene dose) and TK activity Subsequent studies demonstrated that the increase in TK was not a salvage response The experimental data that backs up this contention is provided in the accompanying paper that has been submitted for publication

The inventors proceeded to determine if this information would be useful within the context of cervical carcinoma developing in women who are HIV-positive and are taking AZT to delay the onset of AIDS AZT, a potent inhibitor of HIV replication in vitro and in vivo, requires cellular TK for conversion to AZT- monophosphate (-MP) before other cellular kinases convert AZT-MP to AZT-DP and

its therapeutically active form, AZT-TP (Broder, 1990, Broder et al, 1990, Yarchoan and Broder, 1989) At therapeutic doses in humans, the concentrations of AZT-TP achieved in vivo is not as toxic to human cells as it is to HIV Therefore, the hypothesis that AZT would be phosphorylated to a greater extent and exhibit a greater cell kill in sense cells was tested The degree of phosphorylation of AZT in the 3 cell lines when exposed to 50 μM [methyl- ³ H]AZT for 3 h was first determined As shown in Table 7, AZT-MP and AZT-DP were similar in nontransduced and antisense cells However, sense cells had a 1 6- and 2-fold greater AZT-MP and AZT-DP, respectively

Table 7 Phosphorylation of AZT in Untransduced, Sense, and Antisense Cells

Concentration of AZT Phosphates (pmol/10 ⁶ cells) Cell Lines AZT AZT-MP AZT-DP

1 Untransduced cells 41 3 ± 6.6 194 7 ± 0 7 1 3 ± 0 3

2 Sense cells 55 2 ± 2 9 311 0 ± 2 5* 2 7 ± 0.3

3 Antisense cells 34 1 ± 7.3 180 0 ± 17 5 1 1 ± 0 1

* Significantly different compared to untransduced cells (p<0 005)

Based on the above studies, it may be concluded that sense cells which phosphorylated more AZT would be more sensitive to the cytotoxic effects of AZT Earlier, it was shown that despite the lower proliferation of sense cells, the colony forming efficiency among sense, antisense and untransduced cells was similar (Sun et al 1995) When exposed to AZT, the inhibition profile for colony formation for all 3 cell lines was between 36 5 μM and -365 μM (colony numbers/plate at day 10) However, sense cells showed no colonies at 36.5 μM indicating their greater sensitivity to AZT whereas antisense or untransduced cells were the same as controls not exposed to AZT Only after doubling the concentration of AZT was there evidence for reduction in colony formation with antisense cells In addition, the IC ₅ o (the concentration of AZT at which colony formation was reduced by 50%) was lowest for

sense cells when compared to untransduced or antisense cells (Table 8). Thus, sense cells appeared most susceptible to cytotoxic effects of AZT

Table 8 Cytotoxicity of AZT and Methotrexate to Untransduced,

Sense, and Antisense Cells, as analyzed by Colony Forming Efficiency ¹

Cell Lines AZT ² Methotrexate ²

_

Untransduced 87,000

Sense Transduced 26,500 >6600

Antisense Transduced 55,000 45

¹ - Approximately 500 cells in 3 ml of media were added to 60 X 15 mm culture dishes an were exposed to increasing concentrations of buffer (controls), AZT or MTX. After 10 days, plates were washed, fixed, stained, and colony numbers were enumerated.

² - IC50 (concentration expressed in nM)

To examine effects of these drugs on proliferation of untransduced, sense and antisense cells, an established cytotoxicity assay using cells grown in 96-well tissue culture plates (Cory et al, 1991) was employed. First, the quantity of formazan in the media (at OD ₄₅₀ ) was examined to determine if it was directly proportional to number of living cells in culture. The results revealed a linear relationship of absorbance with cell number over 4 days in culture for all 3 cell lines. Therefore, studies to assess the sensitivity of all 3 cell liens to the cytotoxic effects of increasing doses of AZT were conducted. However, despite the use of 100 μM of AZT, the IC ₅₀ could not be reached for all 3 cells lines. Nevertheless, the inhibition profiles indicated that sense cells were relatively more sensitive at all concentrations of AZT studied.

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 μM 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.

EXAMPLE 4: Role for FR in Cell Proliferation

Although 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. When compared to untransduced cells, sense- and antisense-FR cDNA-transduced cells exhibited an increase and decrease in both FR mRNA and FR expression on the cell surface, respectively. However, when compared to antisense FR cDNA-transduced and untransduced cells, sense FR cDNA-transduced cells exhibited statistically significant (i) increases in total FR, (ii) smaller colonies, (iii) lowered cell proliferation in vitro, and (iv) less tumor volumes with dramatic prolongation of tumor doubling times (225.6 h versus 96 h) following transplantation into nude mice. Finally, (v) using single cell-derived transduced clones, an inverse relationship between cell proliferation and FR expression was established (r = 0.90, / θ.001). Thus, transduction of sense

FR cDNA into cervical carcinoma cells modulated expression of FR and impacted on cell proliferation in vitro and in vivo.

Transfection of FR-α cDNA into various cells that do not constitutively express FR (Matsue et al, 1992; Luhrs et al, 1992; Chung et al, 1993; Bottero et al,

1993) led to greater proliferation and survival when compared to controls cultured in low extracellular folate concentration. However, it was unclear if these finding were

due to independent effects of a greater concentration of intracellular folates accumulated by FR-α cDNA-transfected cells, that prevented folate-deficient cell death at low extracellular folate concentration, a proliferative signal generated at the level of FR per se, or a combination of both To sort out these possibilities, FR-α 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 (μM) 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 μM extracellular folate concentration ensured passive diffusion of folic acid into cells (Antony, 1992). Recent data indicates an inverse relationship between FR expression and cell proliferation in HeLa-IUi cells in vitro and in vivo (Sun et al, 1995). Thus, an additional effect of overexpression ofthe FR in these cells is a reduction in tumor growth. These studies highlight the important new concept that any perturbed event in FR metabolism can also influence cell proliferation. So during studies intended to determine if FR-MP functions on intact cells to cleave-off FR, then the transduction of FR-MP cDNA in the antisense orientation should lead to less expression of FR-MP, this in turn would lead to an increase in expression of FR which, in turn should lead to slower proliferation of these cells (Sun et al, 1995).

FR which mediate the cellular uptake of folates and antifolates are inversely regulated by the extracellular folate concentration. In 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-α mRNA and trαws-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-α 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. Selective RNase H cleavage following hybridization with an 18-mer antisense oligonucleotide complementary to the 18-base RNA fragment quenched both signals indicating that the 18-base RNA sequence was

the c/s-element The interaction of this c/s-element and cytosolic protein was competed by poly(C), but not by poly(U), homopolymers. Cross-linking studies using UV light and Northwestern blot analysis confirmed that the 18-base -element specifically bound 46-kDa proteins.

Whereas antisense oligos upstream (-134 to -151) and downstream (-80 to -97) of the 18-base c/s-element failed to abolish translation, the 18-mer antisense DNA completely inhibited translation of FR-α mRNA without changing its stability Thus, the interaction of the 18-base RNA c/s-element of FR-α and 46 kD-TF proteins likely have an important role in the translational regulation of FR. In addition, since the 46 kD proteins were widely distributed in cells expressing little to no FR-α (including reticulocyte lysates), these species probably have additional functions that are unrelated to FR translation.

These observations are of major significance in cervical carcinoma cells which primarily regulate FR at the translational level in response to extracellular folates and antifolates. With the availability of 18-mer antisense DNA to quench translation, a new "FR-killer" that could function to deplete cells of FR (and folate) is possible The 18-base antisense DNA to the 18-mer c/s-element in FR mRNA is a new form of genetic intervention that could be employed to generate an antifolate effect

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