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WO2000034524A1 - Compositions and methods relating to a new cell cycle regulating gene - Google Patents

Compositions and methods relating to a new cell cycle regulating gene Download PDF

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Publication number
WO2000034524A1
WO2000034524A1 PCT/US1999/029251 US9929251W WO0034524A1 WO 2000034524 A1 WO2000034524 A1 WO 2000034524A1 US 9929251 W US9929251 W US 9929251W WO 0034524 A1 WO0034524 A1 WO 0034524A1
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Prior art keywords
cnn
cell
gene
peptide
promoter
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PCT/US1999/029251
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French (fr)
Inventor
Thomas C. Kaufman
Timothy L. Megraw
Jeffrey K. Cecil
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Advanced Research And Technology Institute
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Priority to AU31165/00A priority Critical patent/AU3116500A/en
Publication of WO2000034524A1 publication Critical patent/WO2000034524A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity

Definitions

  • the present invention relates generally to the fields of molecular and cellular biology. More particularly, it concerns the area of cell cycle regulation and abnormalities in cellular proliferation.
  • Tumor suppressors are naturally-occurring genes known as "tumor suppressors.” Tumor suppressors thus identified in mammalian systems include the gene coding for proteins designated pi 6, p21, p53 and Rb. Similar genes are known to exist in Drosophila and include dap, rux, warts and dig. All of these genes encode proteins that either have enzymatic activity themselves or act as cofactors for enzymes that control the cell cycle, and some have been shown to inhibit cellular proliferation when provided to the appropriate target cell.
  • Centrosomin a 150 kDa centrosomal protein of Drosophila melanogaster, was first identified as a regulatory target of the homeotic genes. The protein is detected in the centrosomes of dividing cells at all stages of development. During the syncytial cleavage divisions, it is present in the centrosomes. After cellularization, during embryogenesis and imaginal disc development, it is localized to the centrosome only during mitosis, and is redistributed into the cytoplasm during interphase. At mid- oogenesis, it is localized to the minus ends of microtubules in the oocyte.
  • centrosomin initial cloning and molecular characterization of the gene encoding centrosomin revealed the presence of three overlapping, alternatively spliced transcripts.
  • the three resulting mRNA encode very similar polypeptides, differing only at their amino termini, and are designated CNN-1, CNN-2 and CNN-3. All three transcripts share 5 exons. Two of the transcripts are found in somatic tissue, while the third is expressed in testes.
  • centrosomin Mutations in centrosomin have been isolated that result in male and female sterility. Using these sterile alleles, it has been shown that centrosomin is required for the assembly of functional centrosomes during the syncytial cleavage. During the syncytial cleavage divisions, the loss of functional centrosomin from the centrosomes results in dramatic defects in nuclear division and spindle organization. Aberrant division and nuclear fusion can be seen from the early cycles of nuclear divisions. Mutant embryos do not cellularize. Examination of spindle morphology and centrosomal composition suggest that mutant embryos lack fully functional centrosomes. Most of the spindles in mutant animals are anastral. Chromosomes apparently organize spindles in mutant embryos in the absence of functional centrosomes. These results suggest that maternally supplied centrosomin is required for the assembly of functional centrosomes during cleavage.
  • an isolated CNN-4 polypeptide.2 may be vertebrate, mammalian, Drosophila melanogaster, or human CNN-4.
  • the CNN-4 has the sequence of SEQ LD NO:2.
  • isolated peptides having between about 10 and about 50 consecutive residues of CNN-4, for example, about 15, about 20, about 25, about 30, about 35, about 40, or about 50 residues.
  • the peptide may be about 10 to about 100 residues in length, for example about 10, about 15, about 20, about 25, about 30, about 40, or about 50 residues.
  • the peptide may be conjugated to a carrier molecule, for example, KLH or BSA.
  • a monoclonal antibody that binds immunologically to CNN-4.
  • the monoclonal antibody may be species specific, may further comprises a detectable label, which may be selected from a fluorescent label, a chemilluminescent label, a radiolabel and an enzyme.
  • a hybridoma cell that produces a monoclonal antibody that binds immunologically to CNN-4.
  • a polyclonal antisera antibodies of which bind immunologically to CNN-4.
  • an isolated polynucleotide encoding CNN-4, or the complement thereof may be derived from vertebrate, mammalian, Drosophila or human sources.
  • the polynucleotide may have the amino acid sequence of SEQ ID NO:2, and may have the polynucleotide sequence of SEQ LD NO: l, or the complement thereof.
  • the polynucleotide may be genomic DNA, complementary DNA and RNA.
  • the polynucleotide may be an expression cassette comprising a DNA segment encoding CNN-4 and a promoter operably linked to said segment.
  • the expression cassette may further comprise a polyadenylation signal operably linked to said segment, for example, a tissue specific promoter, an inducible promoter or a constitutive promoter.
  • the expression cassette is located in a replicable cloning vector which includes an origin of replication.
  • the replicable vector may be a viral vector selected from the group consisting of retrovirus, adenovirus, herpesvirus, vaccinia virus, polyoma virus and adeno- associated virus.
  • the replicable vector may be packaged in a virus particle or in a liposome.
  • the replicable vector is about 5000 bases, about 10,000 bases, about
  • an isolated oligonucleotide of between about 10 and about 100 bases the sequence of which comprises between about 10 and 100 consecutive bases of SEQ ID NO:l.
  • the oligonucleotide may be about 10 bases, about 15 bases, about 20 bases, about 30 bases, about 40 bases, about 50 bases, about 75 bases, about 100 bases in length.
  • a method for expressing a CNN-4 polypeptide comprising the steps of (i) providing a cell; (ii) introducing into said cell an expression cassette comprising a DNA segment encoding a CNN-4 polypeptide and a promoter operably linked to said segment; and (iii) culturing said cell under conditions permitting expression of CNN-4.
  • a method for identifying a CNN-4-binding protein comprising the steps of (u) providing a CNN-4 polypeptide; (ii) contacting said CNN-4 polypeptide with a sample; and (iii) identifying a polypeptide bound to said CNN-4 polypeptide.
  • the method may comprise a two- hybrid selection system.
  • a method of isolating a CNN-4 related gene comprising the steps of (i) selecting a probe consisting essentially of conserved regions from CNN-4; (ii) hybridizing said probe to RNA or DNA from a candidate cell; and (iii) isolating the hybridizing RNA or DNA.
  • the method may further comprise cloning said hybridizing DNA.
  • a method of selecting a cell comprising the steps of (i) transforming a population of cells with an expression cassette comprising a DNA segment encoding a CNN-4 and a promoter operably linked to said segment; (ii) culturing cells under conditions suitable for the expression of CNN-4; and (iii) selecting a cell that survives step (ii).
  • the expression cassette may be part of a larger polynucleotide further comprising a second gene, for example, where the second gene is flanked by genomic sequences of a target site in said cell, and the CNN-4 gene is not flanked by said genomic sequences.
  • a method for selecting a cell comprising (i) transforming a population of cells with an expression cassette comprising a DNA segment encoding CNN-4 and a promoter operably linked to said segment; (ii) culturing cells under conditions suitable for the expression of CNN-4; and (iii) selecting a cell is killed in step (ii).
  • the promoter may be inducible and the conditions result in the induction of said promoter.
  • the method also may comprise a step, between steps (i) and (ii), of making a replicate culture of transformed cells.
  • the expression cassette may be part of a larger polynucleotide further comprising a second gene, for example, where the second gene is a selectable marker.
  • the selectable marker may confer resistance to neomycin, hygromycin, puromycin, zeocin, mycophenolic acid, histidinol or methotrexate.
  • a method of diagnosing a hyperproliferative disease comprising the steps of (i) obtaining a sample from a subject; and (ii) assaying CNN-4 in cells of said sample.
  • the hyperproliferative disease may be cancer, for example, a cancer of the brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood cancer.
  • the assaying may comprise measuring CNN-4 mRNA levels in said sample, measuring CNN-4 protein levels in said sample, e.g., an immunoassay.
  • the assaying also may comprise characterizing the structure of a CNN-4 polynucleotide in said sample, for example, by sequencing.
  • the method also may further comprise the step of comparing the CNN-4 of said sample with that of a non- diseased sample.
  • a method for altering the hyperproliferative phenotype of a cell comprising the step of contacting the cell with
  • the CNN-4 under conditions permitting the uptake of CNN-4 by said cell.
  • the phenotype may be selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth, metastasis and blocking mitosis.
  • the CNN-4 may be encapsulated in a liposome.
  • a method for altering the phenotype of a hyperproliferative cell comprising the step of introducing into said cell an expression cassette comprising (i) a DNA segment encoding CNN-4 and (ii) a promoter active in cell, wherein said promoter is operably linked to said segment, under conditions permitting the uptake of said expression cassette by said hyperproliferative cell.
  • the phenotype may be selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth metastasis and blocking mitosis, and the expression cassette may be encapsulated in a liposome, may be contained in a viral vector selected from the group consisting of retro virus, adenovirus, adeno-associated virus, vaccinia virus and herpesvirus.
  • the viral vector may be packaged in a viral particle.
  • a method for treating a hyperproliferative disease in a subject comprising the step of introducing into a hyperproliferative cell in said subject a CNN-4 polypeptide
  • a method for treating a hype ⁇ roliferative disease in a subject comprising the step of introducing into a hype ⁇ roliferative cell in said subject an expression cassette comprising (i) a DNA. segment encoding CNN-4 and (ii) a promoter active in cell, wherein said promoter is operably linked to said segment, under conditions permitting the uptake of said nucleic acid by said tumor cell.
  • a non-human transgenic organisms the cells of which comprise a transgenic CNN-4 gene.
  • the transgenic CNN-4 gene may be heterologous or homologous to said organism, may be under the control of a promoter (tissue specific, inducible).
  • the CNN-4 may be a mutant CNN- 4 gene; both endogenous copies of CNN-4 may be dysfunctional; the organism is a vertebrate, mammal, Drosophila, or mouse.
  • the invention provides, a method of screening a candidate substance for proliferative activity comprising the steps of (i) providing a cell lacking functional CNN-4; (ii) contacting said cell with said candidate substance; and (iii) determining the effect of said candidate substance on said cell.
  • the determining may comprise comparing one or more characteristics of the cell in the presence of said candidate substance with characteristics of a cell in the absence of said candidate substance, for example, CNN-4 expression, proliferation, metastasis, contact inhibition, soft agar growth, blocking mitosis, tumor formation, tumor progression and tissue invasion.
  • the cell may be contacted in vitro or in vivo.
  • the present invention provides a method for increasing proliferation of cellular population comprising the step of inhibiting CNN-4 function in at least one cell of said population.
  • the inhibition may be achieved by introducing into a cell of said population an expression cassette comprising (i) a DNA segment encoding at least a portion of a CNN-4 complementary sequence and (ii) a promoter active in cell, wherein said promoter is operably linked to said DNA segment, under conditions permitting the uptake of said nucleic acid by said tumor cell.
  • the DNA segment may be complementary to an exon of CNN-4, an intron of CNN-4, adjacent intronic and exonic regions of CNN-4, a splice junction of CNN-4 or a translation initiation site of CNN-4.
  • the inhibition may be achieved by introducing into a cell of said population an expression cassette comprising (i) a DNA segment encoding a single-chain antibody that binds immunologically to CNN-4 and (ii) a promoter active in cell, wherein said promoter is operably linked to said DNA segment, under conditions permitting the uptake of said nucleic acid by said tumor cell.
  • FIG. 1 Map of the Centrosomin Transcription Units. Splicing patterns for centrosomin (cnn) transcripts: CNN-1 (top); CNN-2 (next to top); CNN-3 (next to bottom); CNN-4 (bottom). Also shown are genomic regions with restriction enzyme sites (E - EcoRl; H - Hin ⁇ T ⁇ , S - Sail).
  • FIG. 2 Map of the Centrosomin Genes, mRNAs and Polypeptides.
  • the present invention arises, in one aspect, from the discovery of a new gene, now designated as CNN-4.
  • This gene is part of a complex transcription unit previously identified in connection with three closely related centrosomin transcripts.
  • FIG. 1 The CNN-4 gene shares three exons with CNN-1, CNN-2 and CNN-3, but includes three unique exons that are spliced out of the centrosomin transcripts.
  • the resulting polypeptide contains regions corresponding to exons lc, 3, 4, 4a, 4b and 4c.
  • CNN-4 plays an important role in cell cycle regulation. More particularly, CNN-4 is capable of blocking cell division. Based on its lack of homology with other cell cycle proteins, it is believed that the mechanism by which this occurs is previously unknown. Because of this function, CNN-4, and related compositions, will find use in a variety of different contexts. For example, it may be useful to block CNN-4 activity in cells in culture in order to increase their viability and longevity. It also may prove useful to block CNN-4 activity in order to increase proliferation of tissues in vivo, for example, in tissues that have been damaged and cannot normally repair themselves by increases in number due to cell division. It also is contemplated that increasing CNN-4 activity will find use in treating various hype ⁇ roliferative diseases, such as cancer. Finally, defects in CNN-4 may lead to hype ⁇ roliferative disorders and, hence, determining the status of the CNN-4 gene product provides a diagnostic tool for such disease states.
  • a cell cycle regulator designated CNN-4 This molecule has been shown to block cell division.
  • the present invention also relates to fragments of the polypeptide that may or may not retain the ability to block cell division. Fragments, including varying portions of the N-terminus (truncations) of the molecule may be generated by genetic engineering of translation stop sites within the coding region (discussed below).
  • treatment of the CNN-4 molecule with proteolytic enzymes, known as proteases can produce a variety of N-terminal, C- terminal and internal fragments. Synthetic production of short peptides also is contemplated.
  • fragments may include contiguous residues of the CNN-4 sequence given in SEQ ID NO:2 of 6, 7, 8, 9, 10, 11, 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).
  • the gene for CNN-4 encodes a 462 amino acid polypeptide.
  • the predicted molecular weight of this molecule is 52259.38 with a resulting pi of 7.50.
  • this molecule may be used as a standard in assays where molecule weight and pi are being examined.
  • CNN-4 Sequence analysis indicates that it is principally coiled coil in nature.
  • the amino and carboxy ends as well as two smaller regions in the interior of the protein have low helical potential.
  • the central domain from amino acid residues 40 to 69, 80 to 126 and 152 to 342 all have robust predictions for forming alpha-helical coils. Included in this general coil structure is the prdiction of a leucine zipper extending from residue 45 to 66 within the first coiled domain mentioned above.
  • the protein also has consensus sites for phosphorylation, myrystilization and glycosylation. The number and postion of these sites is given below.
  • BLAST serches of the genome databases reveal no striking homology to any known proteins.
  • CNN-4 When the present application refers to the function of CNN-4 or "wild-type" activity, it is meant that the molecule in question has the ability to block cell division of a cell.
  • Other phenotypes that may be considered to be regulated by CNN-4 are angiogenesis, adhesion, migration, cell-to-cell signaling, cell growth, cell proliferation, density-dependent growth, anchorage-dependent growth, tumor formation, metastasis and others. Determination of which molecules possess this activity may be achieved using assays familiar to those of skill in the art.
  • Variants of CNN-4 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 function or immunogenic activity, and are exemplified by variants lacking the noncoiled sequences 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 of the 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.
  • amino acids of a protein 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 DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 2, below, shows the codons that encode particular amino acids.
  • 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 &
  • 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).
  • 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, i.e., still obtain a biological functionally equivalent protein.
  • 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.
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent and immunologically equivalent protein.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those that are within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take various of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • peptide imetics 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.
  • 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.
  • 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.
  • fusions include linking of functional domains, such as active sites from enzymes, glycosylation domains, cellular targeting signals or transmembrane regions.
  • One particular fusion of interest would include a deletion construct lacking one or more of the phosphorylation sites of CNN-4 or the leucine zipper motif. Fusion to a polypeptide that can be used for purification of the substrate-CNN-4 complex would serve to isolated the substrate for identification and analysis.
  • 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 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 even HPLC.
  • Certain aspects of the present invention concern the purification, and in particular embodiments, the substantial purification, of an encoded protein or peptide.
  • purified protein or peptide as used herein, is intended to refer to a composition, isolatable from other components, wherein the protein or peptide is purified to any degree relative to its naturally-obtainable state.
  • a purified protein or peptide therefore also refers to a protein or peptide, free from the environment in which it may naturally occur.
  • purified will refer to a protein or peptide composition that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term “substantially purified” is used, this designation will refer to a composition in which the protein or peptide forms the major component of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
  • a preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number.”
  • Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an
  • HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
  • High Performance Liquid Chromatography is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes, or at most an hour. Moreover, only a very small volume of the sample is needed because the particles are so small and close-packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be very great because the bands are so narrow that there is very little dilution of the sample.
  • Gel chromatography is a special type of partition chromatography that is based on molecular size.
  • the theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size.
  • the sole factor determining rate of flow is the size.
  • Gel chromatography is unsu ⁇ assed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adso ⁇ tion, less zone spreading and the elution volume is related in a simple matter to molecular weight.
  • Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction.
  • the column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
  • Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin.
  • Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
  • the matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability.
  • the ligand should be coupled in such a way as to not affect its binding properties.
  • the ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand.
  • affinity chromatography One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below. F. Synthetic Peptides
  • the present invention also describes smaller CNN-4-related peptides for use in various embodiments of the present invention. Because of their relatively small size, the peptides of the 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 inco ⁇ orated herein by reference.
  • Short peptide sequences or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily synthesized and then screened in screening assays designed to identify reactive peptides.
  • recombinant DNA technology may be employed wherein a nucleotide sequence which encodes a peptide of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
  • the present invention also provides for the use of CNN-4 proteins or peptides as antigens for the immunization of animals relating to the production of antibodies. It is envisioned that either CNN-4, 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 are the carriers are keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA).
  • KLH keyhole limpet hemocyannin
  • BSA bovine serum albumin
  • the present invention also provides, in another embodiment, oligo- and polynucleotides genes relating to CNN-4.
  • the gene for Drosophila CNN-4 has been identified, as well as variants thereof.
  • the present invention is not limited in scope to these genes, however, as one of ordinary skill could, using such molecules, and related tools, identify related homologs in various other species (e.g., mouse, rat, rabbit, monkey, gibbon, chimp, ape, baboon, human cow, pig, horse, sheep, dog, cat and other species).
  • CNN-4 gene may contain a variety of different bases and yet still produce a corresponding polypeptide that is functionally indistinguishable, and in some cases structurally, from the fruit fly genes disclosed herein.
  • any reference to a oligo- or polynucleotide should be read as encompassing a host cell containing that molecule and, in some cases, capable of expressing the a related protein product.
  • cells expressing oligo- and polynucleotide 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 CNN-4.
  • A. Polynucleotides Encoding CNN-4 The Drosophila sequences disclosed in SEQ LD NOS: l and 3 are polynucleotides according to the present invention. Polynucleotides, or DNA or RNA segments according to the present invention may encode an entire CNN-4 gene, including or excluding introns, an exon of CNN-4, a domain of CNN-4 that blocks cell division, or any other fragment of the CNN-4 sequences set forth herein.
  • the polynucleotide 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).
  • a cDNA plus a natural intron or an intron derived from another gene such engineered molecules are sometime referred to as "mini-genes.”
  • mini-genes these and other nucleic acids of the present invention may be used as molecular weight standards in, for example, gel electrophoresis.
  • the CNN-4 gene is made up of 6 exons, designated as lc, 3, 4, 4a, 4b and 4c.
  • the lc, 3 and 4 exons are shared with the centrosomin transcripts CNN-1 to -3, but exons 4a-c are not. Rather, these exons arise from an intronic region that is completely spliced out of the other centrosomin transcripts.
  • the entire intron is 1296 bases in length and spliced 4a-c segment is 1009 in length.
  • cDNA is intended to refer to DNA prepared using messenger RNA (mRNA) as template.
  • mRNA messenger RNA
  • the entire spliced coding region of CNN-4 is 1812 nucleotides, and the entire cDNA is 1832 nucleotides.
  • 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.
  • the six exons of CNN-4 are located at the following positions in SEQ LD NO: 1 :
  • SEQ LD NO:3 The genomic sequence from the entire centrosomin region is illustrated by SEQ LD NO:3.
  • the exonic positions are as follows:
  • a given CNN-4 gene from a given species may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein.
  • Table 2 the term “an isolated polynucleotide” refers to a polynucleotide molecule that has been isolated, to some extent, from its natural state, i.e., away from 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 2, below), and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.
  • sequences that have at least about 50%, 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 SEQ LD NO: 1 are encompassed in by the present invention.
  • Sequences that are essentially the same as those set forth in SEQ LD NO:l also may be functionally defined as sequences that are capable of hybridizing to a polynucleotide segment containing the complement of SEQ LD NO:l under standard conditions.
  • the DNA segments of the present invention include those encoding biologically functional equivalent CNN-4 proteins and peptides, as described above. Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded.
  • 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. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques or may be introduced randomly and screened later for the desired function, as described below.
  • the present invention also encompasses DNA segments that are complementary, or essentially complementary, to the sequence set forth in SEQ ID NO:l.
  • Nucleic acid sequences that are "complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementary rules.
  • 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 the nucleic acid segment of SEQ LD NO: 1 under relatively stringent conditions such as those described herein. Such sequences may encode the entire CNN-4 protein or functional or non-functional fragments thereof.
  • 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, 11, 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.
  • oligonucleotides encoding 250, 500, 1000, 1386, 1500, 1812, 1832, 2000, 2500, 3000 or 4000, 5000, 10,000, 12,580 bases and longer are contemplated as well.
  • Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions.
  • hybridization 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.
  • hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KC1, 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 KC1,
  • 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 CNN-4 or, more particularly, homologs of CNN-4 from other species.
  • the target DNA will be a genomic or cDNA library, although screening may involve analysis of RNA molecules.
  • screening may involve analysis of RNA molecules.
  • 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, inco ⁇ orating one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA.
  • Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences which encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed.
  • a primer of about 17 to 25 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the 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.
  • Ln 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 of the mutation-bearing strand.
  • E. coli polymerase I Klenow fragment DNA polymerizing enzymes
  • a heteroduplex is formed wherein one strand encodes the original non-mutated sequence and the second strand bears the desired mutation.
  • This heteroduplex vector is then used to transform appropriate cells, such as E. coli cells, and clones are selected that include recombinant vectors bearing the mut
  • sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not meant to be limiting, as there are other ways in which sequence variants of genes may be obtained.
  • recombinant vectors encoding the desired gene may be treated with mutagenic agents, such as hydroxyl amine, to obtain sequence variants.
  • Antisense treatments are one way of addressing this situation.
  • Antisense technology also may be used to "knock-out" function of CNN-4 in the development of cell lines or transgenic mice for research, diagnostic and screening pu ⁇ oses.
  • Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences.
  • 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.
  • 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
  • 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.
  • ribozyme e.g., ribozyme; see below
  • genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
  • 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.
  • Ribozymes are RNA-protein complexes that cleave nucleic acids in a site- specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et al., 1987; Forster and Symons, 1987).
  • ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cook et al., 1981; Michel and Westhof,
  • Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et al, 1981).
  • U.S. Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
  • sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990).
  • expression vectors are employed to express the CNN-4 polypeptide product, which can then be purified and, for example, be used to vaccinate animals to generate antisera or monoclonal antibody with which further studies may be conducted.
  • the expression vectors are used in 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 of the polypeptide.
  • expression construct is meant to include any type of genetic construct containing a nucleic acid coding for a gene product in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • the transcript may be translated into a protein, but it need not be.
  • expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding a gene of interest.
  • 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.
  • 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 of the gene.
  • promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II.
  • 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.
  • promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the 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.
  • such a promoter might include either a human or viral promoter.
  • the human cytomegalovirus (CMV) immediate early gene promoter the SV40 early promoter, the Rous Sarcoma Virus long terminal repeat, beta actin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest.
  • CMV human cytomegalovirus
  • SV40 early promoter the Rous Sarcoma Virus long terminal repeat
  • beta actin promoter glyceraldehyde-3-phosphate dehydrogenase
  • glyceraldehyde-3-phosphate dehydrogenase glyceraldehyde-3-phosphate dehydrogenase
  • the use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given
  • 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.
  • a cDNA insert where a cDNA insert is employed, one will typically 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.
  • a terminator 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.
  • a cell may be identified and selected in vitro or in vivo by including a marker in the expression construct.
  • markers confer an identifiable change to the cell permitting easy identification of cells containing the expression construct.
  • a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.
  • enzymes such as he ⁇ es simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed.
  • tk simplex virus thymidine kinase
  • CAT chloramphenicol acetyltransferase
  • Lmmunologic 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
  • LRES internal ribosome binding sites
  • LRES elements from two members of the picornavirus family have been described (Pelletier and Sonenberg, 1988), as well an LRES from a mammalian message (Macejak and Sarnow, 1991).
  • LRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an LRES, creating polycistronic messages. By virtue of the LRES 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 LRES 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.
  • Delivery of Expression Vectors There are a number of ways in which expression vectors may introduced into cells.
  • 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).
  • adenovirus expression vector is meant to include those constructs containing adenovirus sequences sufficient to (a) support packaging of the construct and (b) to express a polynucleotide that has been cloned therein. In this context, expression does not require that a polypeptide be synthesized.
  • adenovirus a 36 kb, linear, double- stranded DNA virus, allows substitution of large pieces of adenoviral DNA with foreign sequences up to 7 kb (Grunhaus and Horwitz, 1992).
  • retrovirus the adenoviral infection of host cells does not result in chromosomal integration because adenoviral DNA can replicate in an episomal manner without potential genotoxicity.
  • adenoviruses are structurally stable, and no genome rearrangement has been detected after extensive amplification. Adenovirus can infect virtually all epithelial cells regardless of their cell cycle stage. So far, adenoviral infection appears to be linked only to mild disease such as acute respiratory disease in humans.
  • Adenovirus is particularly suitable for use as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target cell range and high infectivity. Both ends of the viral genome contain 100-200 base pair inverted repeats (ITRs), which are cis elements necessary for viral DNA replication and packaging.
  • ITRs inverted repeats
  • the early (E) and late (L) regions of the genome contain different transcription units that are divided by the onset of viral DNA replication.
  • the El region (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication.
  • MLP major late promoter
  • TPL 5 '-tripartite leader
  • 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.
  • adenovirus 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 E3 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.
  • 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.
  • 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.
  • the preferred helper cell line is 293.
  • Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus.
  • natural cell aggregates are grown by inoculating individual cells into 1 liter siliconized spinner flasks (Techne, Cambridge, UK) containing 100-200 ml of medium. Following stirring at 40 ⁇ m, the cell viability is estimated with trypan blue.
  • Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/1) is employed as follows.
  • the adenovirus may be of any of the 42 different known serotypes or subgroups A-F.
  • Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication- defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
  • the typical vector according to the present invention is replication defective and will not have an adenovirus El region.
  • the position of insertion of the construct within the adenovirus sequences is not critical to the invention.
  • the polynucleotide encoding the gene of interest may also be inserted in lieu of the 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 -10 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).
  • adenovirus 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).
  • a polynucleotide 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.
  • a packaging cell line containing the gag, pol, and env genes but without the LTR and packaging components is constructed (Mann et al, 1983).
  • 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,
  • 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 LI 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).
  • 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 he ⁇ esviruses 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).
  • AAV adeno-associated virus
  • Non-viral methods for the transfer of expression constructs into cultured mammalian cells include calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al, 1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al, 1986; Potter et al, 1984), direct microinjection (Harland and Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982; Fraley et al, 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.
  • the DNA segment encoding the gene of interest may be positioned and expressed at different sites.
  • the nucleic acid encoding the gene may be stably integrated into the genome of the cell. This integration may be in the cognate location and orientation via homologous recombination (gene replacement) or it may be integrated in a random, non-specific location (gene augmentation).
  • the nucleic acid may be stably maintained in the cell as a separate, episomal segment of DNA. Such nucleic acid segments or "episomes" encode sequences sufficient to permit maintenance and replication independent of or in synchronization with the host cell cycle. How the expression construct is delivered to a cell and where in the cell the nucleic acid remains is dependent on the type of expression construct employed.
  • the expression construct may simply consist of naked recombinant DNA or plasmids. Transfer of the construct may be performed by any of the methods mentioned above which physically or chemically permeabilize the cell membrane. This is particularly applicable for transfer in vitro but it may be applied to in vivo use as well.
  • Dubensky et al. (1984) successfully injected polyomavirus DNA in the form of calcium phosphate precipitates into liver and spleen of adult and newborn mice demonstrating active viral replication and acute infection. Benvenisty and Neshif (1986) also demonstrated that direct intraperitoneal injection of calcium phosphate-precipitated plasmids results in expression of the transfected genes. It is envisioned that DNA encoding a gene of interest may also be transferred in a similar manner in vivo and express the gene product.
  • 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 inco ⁇ orated by the present invention.
  • the expression construct may be entrapped in a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). Also contemplated are lipofectamine-DNA complexes.
  • the liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda et al, 1989).
  • the liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al, 1991).
  • HMG-1 nuclear non-histone chromosomal proteins
  • the liposome may be complexed or employed in conjunction with both HVJ and HMG-1.
  • expression constructs have been successfully employed in transfer and expression of nucleic acid in vitro and in vivo, then they are applicable for the present invention.
  • a bacterial promoter is employed in the DNA construct, it also will be desirable to include within the liposome an appropriate bacterial polymerase.
  • receptor-mediated delivery vehicles which can be employed to deliver a nucleic acid encoding a particular gene into cells. These take advantage of the selective uptake of macromolecules by receptor-mediated endocytosis in almost all eukaryotic cells. Because of the cell type-specific distribution of various receptors, the delivery can be highly specific (Wu and Wu,
  • Receptor-mediated gene targeting vehicles generally consist of two components: a cell receptor-specific ligand and a DNA-binding agent.
  • ligands have been used for receptor-mediated gene transfer. The most extensively characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and transferrin (Wagner et al, 1990).
  • ASOR asialoorosomucoid
  • transferrin Wang and Wu, 1990
  • the delivery vehicle may comprise a ligand and a liposome.
  • a ligand and a liposome For example, Nicolau et al, (1987) employed lactosyl-ceramide, a galactose-terminal asialganglioside, inco ⁇ orated into liposomes and observed an increase in the uptake of the insulin gene by hepatocytes.
  • a nucleic acid encoding a particular gene also may be specifically delivered into a cell type such as lung, epithelial or tumor cells, by any number of receptor-ligand systems with or without liposomes.
  • epidermal growth factor may be used as the receptor for mediated delivery of a nucleic acid encoding a gene in many tumor cells that exhibit upregulation of EGF receptor.
  • Mannose can be used to target the mannose receptor on liver cells.
  • CD25 T-cell leukemia
  • MAA melanoma
  • 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 of the 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.
  • Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented and are disclosed herein by reference (Freshner, 1992).
  • One embodiment of the foregoing involves the blocking of CNN-4 expression to immortalize cells, for example, in the production of a selected polypeptide.
  • useful mammalian host cell lines are Vero and HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, NLH3T3, RLN and
  • a host cell strain may be chosen that modulates the expression of inserted sequences, or modifies and process the selected product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the correct modification and processing of the foreign protein expressed.
  • Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth).
  • Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products.
  • suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent T-cells.
  • the airlift reactor also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture.
  • the gas stream enters a riser section of the reactor and drives circulation.
  • the airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.
  • the antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation.
  • Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein.
  • For the isolation of membrane proteins cells must be solubilized into detergent micelles.
  • Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations.
  • Antibodies are and their uses are discussed further, below.
  • the present invention contemplates an antibody that is immunoreactive with a CNN-4 polypeptide 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., Harlowe and Lane, 1988).
  • a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal.
  • an immunogen comprising a polypeptide of the present invention
  • a wide range of animal species can be used for the production of antisera.
  • an animal used for production of anti-antisera is a non-human animal including rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.
  • Antibodies both polyclonal and monoclonal, specific for isoforms of antigen may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art.
  • a composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention.
  • Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.
  • the monoclonal antibodies of the present invention will find useful application in standard immunochemical procedures, such as ⁇ LISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to CNN-4-related antigen epitopes. Additionally, it is proposed that monoclonal antibodies specific to the particular CNN-4 of different species may be utilized in other useful applications
  • both polyclonal and monoclonal antibodies against CNN-4 may be used in a variety of embodiments.
  • they may be employed in antibody cloning protocols to obtain cDNAs or genes related to CNN-4. They also may be used in inhibition studies to analyze the effects of CNN-4 related peptides in cells or animals.
  • CNN-4 antibodies will also be useful in immunolocalization studies to analyze the distribution of CNN-4 during various cellular events, for example, to determine the cellular or tissue-specific distribution of CNN-4 polypeptides under different points in the cell cycle.
  • a particularly useful application of such antibodies is in purifying native or recombinant CNN-4, 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 of the present disclosure.
  • 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.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers.
  • Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.
  • the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants.
  • adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.
  • the amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen
  • polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.
  • MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Patent 4,196,265, inco ⁇ orated herein by reference.
  • this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified CNN-4 protein, polypeptide or peptide or cell expressing high levels of CNN-4.
  • the immunizing composition is administered in a manner effective to stimulate antibody producing cells.
  • Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep frog cells is also possible.
  • the use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.
  • somatic cells with the potential for producing antibodies, specifically B-lymphocytes (B-cells), are selected for use in the mAb generating protocol.
  • B-cells B-lymphocytes
  • These cells may be obtained from biopsied spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • a panel of animals will have been immunized and the spleen of animal with the highest antibody titer will be removed and the spleen lymphocytes obtained by homogenizing the spleen with a syringe.
  • a spleen from an immunized mouse contains approximately 5 x 10 7 to 2 x 10 8 lymphocytes.
  • the antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized.
  • Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
  • any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 1986; Campbell, 1984).
  • the immunized animal is a mouse
  • rats one may use R210.RCY3, Y3-Ag 1.2.3, LR983F 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).
  • PEG polyethylene glycol
  • the use of electrically induced fusion methods is also appropriate (Goding, 1986).
  • the selective medium is generally one that contains an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • agents are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, whereas azaserine blocks only purine synthesis.
  • the media is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium).
  • HAT medium a source of nucleotides
  • azaserine the media is supplemented with hypoxanthine.
  • the preferred selection medium is HAT. Only cells capable of operating nucleotide salvage pathways are able to survive in HAT medium.
  • the myeloma cells are defective in key enzymes of the salvage pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.
  • HPRT hypoxanthine phosphoribosyl transferase
  • the B-cells can operate this pathway, but they have a limited life span in culture and generally die within about two weeks. Therefore, the only cells that can survive in the selective media are those hybrids formed from myeloma and B-cells.
  • This culturing provides a population of hybridomas from which specific hybridomas are selected.
  • selection of hybridomas is performed by culturing the cells by single-clone dilution in microtiter plates, followed by testing the individual clonal supematants (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. V. Methods for Screening of Active Compounds
  • the present invention also contemplates the use of CNN-4 and active fragments, and polynucleotides coding therefor, in the screening of compounds for activity in either stimulating CNN-4 activity, overcoming the lack of CNN-4, blocking the effect of CNN-4, or overcoming the effects of a mutant CNN-4.
  • These assays may make use of a variety of different formats and may depend on the kind of "activity" for which the screen is being conducted.
  • Contemplated functional "read-outs” include binding to a compound, inhibition of binding to a substrate, ligand, receptor or other binding partner by a compound, inhibition or stimulation of cell-to-cell signaling, growth, metastasis, cell division, cell migration, soft agar colony formation, contact inhibition, tissue invasiveness, angiogenesis, apoptosis, tumor establishment or progression or other malignant phenotype.
  • the term "candidate substance” may be a protein or fragment thereof, a small molecule inhibitor, an oligo- or polynucleotide, of natural or synthetic origin. It may prove to be the case that the most useful pharmacological compounds for identification through application of the screening assay will be compounds that are structurally related to CNN-4 or targets of CNN-4.
  • the active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive.
  • the active compounds may include fragments or parts of naturally-occurring compounds or may be found as active combinations of known compounds which are otherwise inactive. Accordingly, the present invention provides screening assays to identify agents from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be polypeptide or fragment thereof, oligonucleotide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design. "Effective amounts" in certain circumstances are those amounts effective to reproducibly produce the assayed result.
  • the invention is to be applied for the screening of compounds that bind to the CNN-4 polypeptide or fragment thereof.
  • the polypeptide or fragment may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the polypeptide or the compound may be labeled, thereby permitting determining of binding.
  • the assay may measure the inhibition of binding of CNN-4 to a natural or artificial substrate or binding partner.
  • Competitive binding assays can be performed in which one of the agents (CNN-4, binding partner or compound) is labeled.
  • the polypeptide will be the labeled species.
  • One may measure the amount of free label versus bound label to determine binding or inhibition of binding.
  • Purified CNN-4 can be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase.
  • fusion proteins containing a reactive region may be used to link the CNN-4 active region to a solid phase.
  • Various cell lines containing wild-type CNN-4, or natural or engineered mutations in CNN-4 can be used to study various functional attributes of CNN-4 and how a candidate compound affects these attributes. Methods for engineering mutations are described elsewhere in this document.
  • the compound would be formulated appropriately, given its biochemical nature, and contacted with a target cell.
  • culture may be required.
  • the cell may then be examined by virtue of a number of different physiologic assays.
  • molecular analysis may be performed in which the function of CNN-4, or related pathways, may be explored. This may involve assays such as those for protein expression, protein function, substrate utilization, etc.
  • the present invention also encompasses the use of various whole organism models. By developing or isolating mutant cells lines that fail to express normal CNN-4, one can generate cancer models in mice that will be highly predictive of cancers in humans and other mammals. These models may employ the orthotopic or systemic administration of tumor cells to mimic primary and or metastatic cancers.
  • transgenic animals that lack a wild-type CNN-4 may be utilized as models for cancer development and treatment.
  • Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal.
  • Administration will be by any route the could be utilized for clinical or non-clinical pu ⁇ oses, including but not limited to oral, nasal, buccal, rectal, vaginal or topical.
  • administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection.
  • systemic intravenous injection regional administration via blood or lymph supply and intratumoral injection.
  • Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Such criteria include, but are not limited to, survival, reduction of tumor burden or mass, arrest or slowing of tumor progression, elimination of tumors, inhibition or prevention of metastasis, increased activity level, improvement in immune effector function and improved food intake.
  • Rational Drug Design The goal of rational drug design is to produce structural analogs of biologically active polypeptides or compounds with which they interact (agonists, antagonists, inhibitors, binding partners, etc.). By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for
  • CNN-4 or a fragment thereof. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches.
  • CNN-4 may design molecules which have improved CNN-4 activity or which act as stimulators, inhibitors, agonists, antagonists of CNN-4 or molecules affected by CNN-4 function.
  • sufficient amounts of CNN-4 can be produced to perform crystallographic studies.
  • knowledge of the polypeptide sequences permits computer employed predictions of structure-function relationships.
  • the ability to immortalize certain cell types can have a significant benefit. For example, manipulation of certain cells that are hard to maintain in culture over long periods of time (lymphocytes) and cells that can be immortalized, but lose their primary cell characteristics (pancreatic ⁇ -cells), may be improved by blocking CNN-4 function.
  • the first method involves the use of a pharmaceuticals.
  • the second method involves the use of an antisense construct designed to inhibit CNN-4 transcription, splicing, or translation. This aspect is described in greater detail above.
  • the third method involves a CNN-4 directed ribozyme, also as described above.
  • the fourth method involves use of a genetic construct encoding for a single chain antibody directed to CNN-4. This embodiment is discussed further, below.
  • a single-chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule.
  • Single-chain antibody variable fragments scFvs in which the C-terminus of one variable domain is tethered to the N-terminus of the other via a 15 to 25 amino acid peptide or linker, have been developed without significantly disrupting antigen binding or specificity of the binding (Bedzyk et al., 1990; Chaudhary et al., 1990). These Fvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody.
  • Single-chain antibodies can be synthesized in a host cell, targeted to particular cellular compartments, and used to interfere, in a highly specific manner, with cell growth and metabolism (Richardson and Marasco, 1995). Recently, single-chain antibodies were utilized for the phenotypic knockout of growth-factor receptors, the functional inactivation of p21r ⁇ s, and the inhibition of HIV-1 replication. Intracellular antibodies offer a simple and effective alternative to other forms of gene inactivation, as well as demonstrate a clear potential as reagents for cancer therapy and for the control of infectious diseases. Single-chain antigen-binding proteins also represent potentially unique molecules for targeted delivery of drugs, toxins, or radionuclides to a tumor site, and show increased accessibility to tumor cells in vivo (Yokoda et al, 1992).
  • CNN-4 Given CNN-4' s involvement with cell cycle regulation, it is proposed that some hype ⁇ roliferative diseases may be caused by aberrations in this gene. Therefore, CNN-4 and the corresponding gene may be targeted in diagnostic or prognostic indications of such disease. More specifically, point mutations, deletions, insertions or regulatory pertubations relating to CNN-4 may cause or promote hype ⁇ roliferative diseases such as cancers. Other phenomena associated with malignancy that may be affected by CNN-4 expression include metastasis, angiogenesis and tissue invasion.
  • One embodiment of the instant invention comprises a method for detecting variation in the expression of CNN-4. This may comprises determining that level of CNN-4 or determining specific alterations in the expressed product. This sort of assay has importance in the diagnosis of hype ⁇ roliferative diseases, including cancers.
  • Such cancers may involve cancers of the brain, lung, liver, spleen, kidney, pancreas, small intestine, blood cells, lymph node, colon, breast, endometrium, stomach, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue.
  • the 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.
  • 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.
  • the specific polynucleotide of interest is identified in the sample directly using amplification or with a second, known oligo- or polynucleotide following amplification.
  • the identified product is detected.
  • the detection may be performed by visual means (e.g., ethidium bromide staining of a gel).
  • the detection may involve indirect identification of the product via chemilluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; Bellus, 1994).
  • a cell takes a genetic step toward oncogenic transformation when one allele of a cell cycle regulatory gene is inactivated due to inheritance of a germline lesion or acquisition of a somatic mutation.
  • the inactivation of the other allele of the gene usually involves a somatic micromutation or chromosomal allelic deletion that results in loss of heterozygosity (LOH).
  • LH heterozygosity
  • both copies of a gene may be lost by homozygous deletion.
  • FISH fluorescent in situ hybridization
  • PFGE direct DNA sequencing
  • Southern or Northern blotting single-stranded conformation analysis
  • SSCA RNAse protection assay
  • ASO allele-specific oligonucleotide
  • dot blot analysis denaturing gradient gel electrophoresis, RFLP and PCRTM-SSCP.
  • primer is meant to encompass any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template- dependent process.
  • primers are oligonucleotides from ten to twenty base pairs in length, but longer sequences can be employed.
  • Primers may be provided in double-stranded or single-stranded form, although the single-stranded form is preferred.
  • Probes are defined differently, although they may act as primers. Probes, while perhaps capable of priming, are designed to binding to the target DNA or RNA and need not be used in an amplification process.
  • the probes or primers are labeled with radioactive species ( 32 P, 14 C, 35 S, 3 H, or other label), with a fluorophore (rhodamine, fluorescein) or a chemillumiscent (luciferase).
  • radioactive species 32 P, 14 C, 35 S, 3 H, or other label
  • fluorophore rhodamine, fluorescein
  • chemillumiscent luciferase
  • PCRTM polymerase chain reaction
  • 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 primers will bind to the marker and the polymerase will cause the primers to be extended along the marker sequence by adding on nucleotides.
  • the extended primers will dissociate from the marker to form reaction products, excess primers will bind to the marker and to the reaction products and the process is repeated.
  • a reverse transcriptase PCR amplification procedure may be performed in order to quantify the amount of mRNA amplified.
  • RNA into cDNA are well known and described in Sambrook et al, 1989.
  • Alternative methods for reverse transcription utilize thermostable, RNA-dependent DNA polymerases. These methods are described in WO 90/07641 filed December 21, 1990. Polymerase chain reaction methodologies are well known in the art.
  • LCR ligase chain reaction
  • Qbeta Replicase described in PCT Application No. PCT/US87/00880, may 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 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)
  • RCR Repair Chain Reaction
  • annealing several probes throughout a region targeted for amplification followed by a repair reaction in which only two of the four bases are present.
  • Target specific sequences can also be detected using a cyclic probe reaction (CPR).
  • CPR cyclic probe reaction
  • Still another amplification methods described in GB Application No. 2 202 328, and in PCT Application No. PCT/US 89/01025, each of which is inco ⁇ orated 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, inco ⁇ orated herein by reference in their entirety). Davey et al, EPO No.
  • 329 822 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.
  • ssRNA single-stranded RNA
  • dsDNA double-stranded DNA
  • Miller et al, PCT Application WO 89/06700 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 transcription of many RNA copies of the sequence.
  • amplification methods include “RACE” and “one-sided PCR TM” (Frohman, M.A., In: RCR TM PROTOCOLS: A GUIDE TO METHODS AND APPLICATIONS, Academic Press, N.Y., 1990; Ohara et al, 1989; each herein inco ⁇ orated by reference in their entirety).
  • Methods based on ligation of two (or more) oligonucleotides in the presence of a polynucleotide 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), inco ⁇ orated herein by reference in its entirety.
  • Southern/Northern Blotting Blotting techniques are well known to those of skill in the art. Southern blotting involves the use of DNA as a target, whereas Northern blotting involves the use of RNA as a target. Each provide different types of information, although cDNA blotting is analogous, in many aspects, to blotting or RNA species.
  • a probe is used to target a DNA or RNA species that has been immobilized on a suitable matrix, often a filter of nitrocellulose.
  • the different species should be spatially separated to facilitate analysis. This often is accomplished by gel electrophoresis of nucleic acid species followed by "blotting" on to the filter. 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.
  • amplification products are separated by agarose, agarose-acrylamide or polyacrylamide gel electrophoresis using standard methods. See Sambrook et al, 1989.
  • chromatographic techniques may be employed to effect separation.
  • chromatography There are many kinds of chromatography which may be used in the present invention: adso ⁇ tion, partition, ion-exchange and molecular sieve, and many specialized techniques for using them including column, paper, thin-layer and gas chromatography (Freifelder, 1982).
  • Products may be visualized in order to confirm amplification of the marker sequences.
  • One typical visualization method involves staining of a gel with ethidium bromide and visualization under UV light.
  • the amplification products can then be exposed to x-ray film or visualized under the appropriate stimulating spectra, following separation.
  • visualization is achieved indirectly.
  • a labeled nucleic acid probe is brought into contact with the amplified marker sequence.
  • the probe preferably is conjugated to a chromophore but may be radiolabeled.
  • the probe is conjugated to a binding partner, such as an antibody or biotin, and the other member of the binding pair carries a detectable moiety.
  • 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.
  • amplification products described above may be subjected to sequence analysis to identify specific kinds of variations using standard sequence analysis techniques.
  • 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.
  • oligonucleotide primers may be designed to permit the amplification of sequences throughout the CNN-4 gene that may then be analyzed by direct sequencing.
  • Kit Components All the essential materials and reagents required for detecting and sequencing
  • CNN-4 and variants thereof may be assembled together in a kit.
  • This generally will comprise preselected primers and probes.
  • enzymes suitable for amplifying nucleic acids including various polymerases (RT, Taq, Sequenase etc.), deoxynucleotides and buffers to provide the necessary reaction mixture for amplification.
  • kits also generally will comprise, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe.
  • RT Reverse transcription
  • RT-PCRTM relative quantitative PCRTM
  • PCR the number of molecules of the amplified target DNA increase by a factor approaching two with every cycle of the reaction until some reagent becomes limiting. Thereafter, the rate of amplification becomes increasingly diminished until there is no increase in the amplified target between cycles.
  • a graph is plotted in which the cycle number is on the X axis and the log of the concentration of the amplified target DNA is on the Y axis, a curved line of characteristic shape is formed by connecting the plotted points. Beginning with the first cycle, the slope of the line is positive and constant. This is said to be the linear portion of the curve. After a reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.
  • the concentration of the target DNA in the linear portion of the PCR amplification is directly proportional to the starting concentration of the target before the reaction began.
  • concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundances of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundances is only true in the linear range of the PCR reaction.
  • the final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the first condition that must be met before the relative abundances of a mRNA species can be determined by RT-PCR for a collection of RNA populations is that the concentrations of the amplified PCR products must be sampled when the PCRTM reactions are in the linear portion of their curves.
  • the second condition that must be met for an RT-PCR TM experiment to successfully determine the relative abundances of a particular mRNA species is that relative concentrations of the amplifiable cDNAs must be normalized to some independent standard.
  • the goal of an RT-PCR experiment is to determine the abundance of a particular mRNA species relative to the average abundance of all mRNA species in the sample.
  • mRNAs for ⁇ - actin, asparagine synthetase and lipocortin ⁇ were used as external and internal standards to which the relative abundance of other mRNAs are compared.
  • RT-PCR is performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable cDNA fragment that is larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5-100 fold higher than the mRNA encoding the target.
  • This assay measures relative abundance, not absolute abundance of the respective mRNA species.
  • Chip Technologies Specifically contemplated by the present inventors are chip-based DNA technologies such as those described by Hacia et al (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al. (1994); Fodor et al. (1991).
  • Antibodies of the present invention can be used in characterizing the CNN-4 content of healthy and diseased tissues, through techniques such as ELISAs and
  • anti-CNN-4 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 adso ⁇ tion sites on the immobilizing surface and thus reduces the background caused by non-specific binding of antigen onto the surface.
  • BSA bovine serum albumin
  • the immobilizing surface After binding of antibody to the well, coating with a non-reactive material to reduce background, and washing to remove unbound material, the immobilizing surface is contacted with the sample to be tested in a manner conducive to immune complex (antigen antibody) formation. 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 immunoaffinity for CNN-4 that differs the first antibody.
  • Appropriate conditions preferably include diluting the sample with diluents such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween ® . These added agents also tend to assist in the reduction of nonspecific background.
  • BSA bovine gamma globulin
  • PBS phosphate buffered saline
  • 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.
  • the second antibody will preferably have an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • an associated enzyme that will generate a color development upon incubating with an appropriate chromogenic substrate.
  • one will desire to contact and incubate the second antibody-bound surface with a urease or peroxidase-conjugated anti-human IgG for a period of time and under conditions which favor the development of immunocomplex formation (e.g., incubation for 2 hr at room temperature in a PBS -containing solution such as PBS/Tween ® ).
  • the amount of label is quantified by incubation with a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label. Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • a chromogenic substrate such as urea and bromocresol pu ⁇ le or 2,2'-azino-di-(3- ethyl-benzthiazoline)-6-sulfonic acid (ABTS) and H 2 O 2 , in the case of peroxidase as the enzyme label.
  • Quantitation is then achieved by measuring the degree of color generation, e.g., using a visible spectrum spectrophotometer.
  • the preceding format may be altered by first binding the sample to the assay plate. Then, primary antibody is incubated with the assay plate, followed by detecting of bound primary antibody using a labeled second antibody with specificity for the primary antibody.
  • the antibody compositions of the present invention will find great use in immunoblot or Western blot analysis.
  • the antibodies may be used as high- affinity primary reagents for the identification of proteins immobilized onto a solid support matrix, such as nitrocellulose, nylon or combinations thereof.
  • a solid support matrix such as nitrocellulose, nylon or combinations thereof.
  • immunoprecipitation followed by gel electrophoresis, these may be used as a single step reagent for use in detecting antigens against which secondary reagents used in the detection of the antigen cause an adverse background.
  • Immunologically-based detection methods for use in conjunction with Western blotting include enzymatically- , radiolabel-, or fluorescently-tagged secondary antibodies against the toxin moiety are considered to be of particular use in this regard.
  • the present invention also involves, in another embodiment, the treatment of hype ⁇ roliferative diseases.
  • Virtually any hype ⁇ roliferative disease could be treated; it is not a requirement that CNN-4 be mutated or abnormal. Rather, the overexpression of CNN-4 may actually overcome other defects within the cell.
  • a wide variety of hype ⁇ roliferative diseases may be treated using CNN-4 therapy, including cancers of the brain, lung, liver, spleen, kidney, lymph node, pancreas, small intestine, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue.
  • One of the therapeutic embodiments contemplated by the present inventors is the intervention, at the molecular level, in the events involved in the tumorigenesis of some cancers.
  • the present inventors intend to provide, to a cancer cell, an expression construct capable of providing CNN-4 to that cell.
  • an expression construct capable of providing CNN-4 to that cell.
  • viral vectors such as adenovirus, adeno-associated virus, he ⁇ esvirus, vaccinia virus and retrovirus.
  • liposomally-encapsulated expression vector are also preferred.
  • routes are contemplated for various diseases.
  • the section below on routes contains an extensive list of possible routes.
  • systemic delivery is contemplated. This will prove especially important for attacking microscopic or metastatic cancer.
  • discrete tissue mass may be identified, a variety of direct, local and regional approaches may be taken.
  • the tumor may be directly injected with the expression vector.
  • a tumor bed may be treated prior to, during or after resection. Following resection, one generally will deliver the vector by a catheter left in place following surgery.
  • One may utilize the tumor vasculature to introduce the vector into the tumor by injecting a supporting vein or artery.
  • a more distal blood supply route also may be utilized.
  • ex vivo gene therapy is contemplated. This approach is particularly suited, although not limited, to treatment of bone marrow associated diseases.
  • cells from the patient are removed and maintained outside the body for at least some period of time. During this period, a therapy is delivered, after which the cells are reintroduced into the patient; hopefully, any hype ⁇ roliferative cells in the sample have been killed.
  • ABMT Autologous bone marrow transplant
  • the notion behind ABMT is that the patient will serve as his or her own bone marrow donor.
  • a normally lethal dose of irradiation or chemotherapeutic may be delivered to the patient to kill tumor cells, and the bone marrow repopulated with the patients own cells that have been maintained (and perhaps expanded) ex vivo.
  • irradiation or chemotherapeutic may be delivered to the patient to kill tumor cells, and the bone marrow repopulated with the patients own cells that have been maintained (and perhaps expanded) ex vivo.
  • Use of gene therapy to accomplish this goal is yet another way CNN-4 may be utilized according to the present invention.
  • Immunotherapeutics generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells.
  • the immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell.
  • the antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing.
  • the antibody also may be conjugated to a drug or toxin
  • the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target.
  • Various effector cells include cytotoxic T cells and NK cells.
  • Another therapy approach is the provision, to a subject, of CNN-4 polypeptide, active fragments, synthetic peptides, mimetics or other analogs thereof.
  • the protein may be produced by recombinant expression means or, if small enough, generated by an automated peptide synthesizer.
  • Formulations would be selected based on the route of administration and pu ⁇ ose including, but not limited to, liposomal formulations and classic pharmaceutical preparations.
  • compositions of the present invention To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the hype ⁇ roliferative phenotype of cells, using the methods and compositions of the present invention, one may contact a "target" cell with a CNN-4 expression construct and at least one other agent. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the expression construct and the agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the agent.
  • the gene therapy treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks to months.
  • the other agent and expression construct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell.
  • CNN-4 is "A” and the other agent is "B”, as exemplified below: A/B/A B/A B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
  • both agents are delivered to a cell in a combined amount effective to kill the cell.
  • Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell.
  • Such agents and factors include radiation and waves that induce DNA damage such as, ⁇ -irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like.
  • Chemotherapeutic agents contemplated to be of use include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, and cisplatin (CDDP).
  • the invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide.
  • the use of cisplatin in combination with a CNN-4 expression construct is particularly preferred as this compound.
  • the tumor cells In treating cancer according to the invention, one would contact the tumor cells with an agent in addition to the expression construct. This may be achieved by irradiating the localized tumor site with radiation such as X-rays, UV-light, ⁇ -rays or even microwaves. Alternatively, the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin.
  • the agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a CNN-4 expression construct, as described above.
  • CNN-4-targeted therapies with chemo- and radiotherapies
  • combination with other gene therapies will be advantageous.
  • targeting of CNN-4 and p53, Rb, p21 or pl ⁇ mutations at the same time may produce an improved anti-cancer treatment.
  • tumor-related gene conceivably can be targeted in this manner, for example, p21, p53, Rb, APC, DCC, NF-1, NF-2, BCRA2, pi 6, FHIT, WT-1, MEN-I, MEN-LI, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf, erb, src, fins, jun, trk, ret, gsp, hst, bcl and abl.
  • the present invention presents the opportunity to intervene therapeutically in clinical states where a lack of cellular growth either is at the cause, or prevents recover from, the state.
  • states include bone wasting disease or damage, nervous tissue degenerative disease or damage, hematopoietic cell disease, and connective tissue healing.
  • CNN-4 gene is inserted into an expression cassette.
  • the CNN-4 express cassette is covalently linked to a gene that the investigator wishes to transfer into a particular cell, which is either under control of the same or a different promoter, as compared to
  • the CNN-4 gene can be used as follows.
  • the CNN-4 gene is placed at one end of a DNA construct that comprises a selected gene to be transferred, flanked by target gene sequences.
  • the CNN-4 gene is under the control of a promoter that will express CNN-4 in the host cell, under suitable conditions, at a level that will kill the cell.
  • a target gene within a host cell is selected as the location into which a selected gene is to be transferred. Sequences homologous to the target gene are included in the expression construct, and the selected gene is inserted such that target gene homologous sequences are interrupted by the selected gene or, put another way, such the target gene homologous sequences "flank" the selected gene. In preferred embodiments, a drug selectable marker gene also is inserted into the target gene homologous sequences. Given this possibility, it should be apparent that the term “flank” is used broadly herein, namely, as describing target homologous sequences that are both upstream (5') and downstream (3') of the selected gene and/or the drug selectable marker gene. In effect, the flanking sequences need not directly abut the genes they "flank.”
  • construct for use in this embodiment is further characterized as having a functional CNN-4 gene attached thereto.
  • one possible arrangement of sequences would be:
  • CNN-4 could come at the 3'-end of the construct and the selected gene and drug-selectable marker genes could exchange positions.
  • a modification of this procedure is one where no selected gene is included, i.e., only the selectable marker is inserted into the target gene homologous sequences.
  • Examples of processes that use negative selection to enrich for homologous recombinants include the disruption of targeted genes in embryonic stem cells or transformed cell lines (Mortensen, 1993, Willnow & Herz, 1994) and the production of recombinant virus such as adenovirus (Imler et al, 1995).
  • the CNN-4 gene can be used as a tool for screening for transfer of expression vectors in a non-homologous fashion.
  • the expression construct contains the selected gene that is to be delivered, a drug selectable marker gene and a CNN-4 cDNA or genomic clone.
  • the selected gene is inte ⁇ osed between the CNN-4 transporter gene and the marker gene.
  • the principle behind the selection process is as follows. While some recombinants may only achieve partial integration of the construct, i.e., the drug selectable marker or the CNN-4 sequences, a cell expressing products of both these vector genes also likely contains the intervening sequences, namely, the selected gene. Thus, following recombination, the cells are screened for drug resistance. Of the remaining cells, these are screened for CNN-4 expression, i.e., death under suitable conditions. If the cell satisfies these double-selection criteria, they should also expression the selected gene.
  • the drug screen is a conventional positive selection protocol where the surviving cells are those desired.
  • the CNN-4 screen may be accomplished by utilizing an inducible promoter to control the expression of CNN-4. In an uninduced state, no CNN-4 is produced. However, using the proper signal, CNN-4 synthesis may be turned no and, hence, cells that contain this gene can be identified. Being a negative selection process, this approach generally involves preparing replica or duplicate cultures, where a first and a second cell culture of the drug-resistant cells are prepared. One cell culture is contacted with the inducer, and cells are identified that are CNN-4 + , one identifies replicas that died under induction. Further characterization of these doubly-selected clones should confirm expression of the selected gene.
  • antibiotic resistance markers examples include those conferring resistance to neomycin, hygromycin, puromycin or zeocin; and xanthine-guanine phosphoribosyl transferase, HisD and dihydrofolate reductase genes.
  • the operation of drug selection or antibiotic resistance marker genes is well known to those of skill in the art.
  • transgenic animals are produced which contain a transgene encoding a functional CNN-4 polypeptide or variants thereof.
  • Transgenic animals expressing CNN-4 transgenes, recombinant cell lines derived from such animals and transgenic embryos may be useful in methods for screening for and identifying agents that induce or repress function of CNN-4.
  • Transgenic animals of the present invention also can be used as models for studying indications such as cancers. In cells where CNN-4 is lethal, it will be desirable to place the CNN-4 gene under the control of an inducible promoter.
  • Selected animals include fruit flies, mice, rats, sheep, guinea pigs, cows or other suitable organism.
  • a CNN-4 transgene is introduced into a non-human host to produce a transgenic animal expressing a CNN-4 product.
  • the transgenic animal is produced by the integration of the transgene into the genome in a manner that permits the expression of the transgene.
  • Methods for producing transgenic animals are generally described by Wagner and Hoppe (U.S. Patent 4,873,191; which is inco ⁇ orated herein by reference), Brinster et al.
  • a CNN-4 gene flanked by genomic sequences is transferred by microinjection into a fertilized egg.
  • the microinjected eggs are implanted into a host female, and the progeny are screened for the expression of the transgene.
  • Transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish.
  • transgenic mice are generated which overexpress CNN-4 (e.g., inducibly) or express a mutant form of the CNN-4 polypeptide.
  • overexpress CNN-4 e.g., inducibly
  • the absence of a CNN-4 in "knock-out" mice permits the study of the effects that loss of CNN-4 protein has on a cell in vivo.
  • Knock-out mice also provide a model for the development of CNN-4-related hype ⁇ roliferative diseases.
  • transgenic animals and cell lines derived from such animals may find use in certain testing experiments.
  • transgenic animals and cell lines capable of expressing wild-type or mutant CNN-4 may be exposed to test substances. These test substances can be screened for the ability to enhance wild-type CNN-4 expression and or function or impair the expression or function of mutant CNN-4.
  • compositions - expression vectors, virus stocks, proteins, antibodies and drugs - in a form appropriate for the intended application.
  • 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.
  • phrases "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.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well know in the art. 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 inco ⁇ orated 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.
  • 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.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial an antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged abso ⁇ tion of the injectable compositions can be brought about by the use in the compositions of agents delaying abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by inco ⁇ orating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by inco ⁇ orating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion 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 inco ⁇ orated into the compositions.
  • the polypeptides of the present invention may be inco ⁇ orated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
  • a mouthwash may be prepared inco ⁇ orating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be inco ⁇ orated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
  • the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
  • the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
  • compositions of the present invention may be formulated in a neutral or salt form.
  • Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
  • solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
  • the solution For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. 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.
  • Graham and van der Eb "A new technique for the assay of infectivity of human adenovirus 5 DNA", Virology, 52:456-467, 1973. Graham et al, "Characteristics of a human cell line transformed by DNA from human adenovirus type 5", J. Gen. Virol, 36:59-72, 1977. Great Britain Patent No. 2 202 328 Grunhaus and Horwitz, "Adenovirus as cloning vector", Seminar in Virology, 3:237-
  • Hermonat and Muzycska "Use of adenoassociated virus as a mammalian DNA cloning vector: Transduction of neomycin resistance into mammalian tissue culture cells", Proc. Nat. Acad. Sci. USA, 81:6466-6470, 1984.
  • Nicolas and Rubinstein In: Vectors: A survey of molecular cloning vectors and their uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp. 494-513,

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Abstract

The present invention is directed to a newly identified gene product, its corresponding gene, and uses thereof. The product, designated CNN-4, is related to Drosophila centrosomin proteins, and has been shown to play an important role in blocking of cell division. Thus, modulating the effects of this molecule permits one to either upregulate cell division, which has important implication for basis research tools as well as in restorative therapy, or to downregulate cellular proliferation, which could be applied in treatment of hyperproliferative diseases such as cancer.

Description

DESCRIPTION
COMPOSITIONS AND METHODS RELATING TO A NEW CELL CYCLE
REGULATING GENE
BACKGROUND OF THE INVENTION
The government may own rights in the present invention pursuant to grant No. 9728694 from the National Science Foundation.
1. Field of the Invention The present invention relates generally to the fields of molecular and cellular biology. More particularly, it concerns the area of cell cycle regulation and abnormalities in cellular proliferation.
2. Description of Related Art Processes involving the regulation of cellular proliferation have long been of interest to cell biologists. In particular, intense research efforts have been directed towards identifying and understanding abnormalities in cell cycle regulation, especially as they apply to hyperproliferative diseases such as cancer. At the heart of this research is the goal of understanding how cell division takes place, and how it can be regulated and/or stopped. Preventing unregulated or uncontrolled division is the primary goal to be achieved in treating cancer and other hyperproliferative disorders.
Many of the reagents currently known to affect cell division, including radiation and classic chemotherapeutics, have widespread and undesirable affects on cellular metabolism. The existence of a normally-occurring, endogenous gene product that can serve to inhibit uncontrolled cellular proliferation, but avoid toxic effects, would be of great interest from a treatment perspective. It also would provide important additional insights on cell cycle function that could be exploited in a wide variety of contexts. As stated above, most radiation and chemical agents exhibit undesired side affects in the hands of the even the most skilled physician. The identification of a naturally occurring product that could avoid these problems is, without question, of paramount importance. Putative candidates as molecular "silver bullets" are naturally-occurring genes known as "tumor suppressors." Tumor suppressors thus identified in mammalian systems include the gene coding for proteins designated pi 6, p21, p53 and Rb. Similar genes are known to exist in Drosophila and include dap, rux, warts and dig. All of these genes encode proteins that either have enzymatic activity themselves or act as cofactors for enzymes that control the cell cycle, and some have been shown to inhibit cellular proliferation when provided to the appropriate target cell.
Centrosomin, a 150 kDa centrosomal protein of Drosophila melanogaster, was first identified as a regulatory target of the homeotic genes. The protein is detected in the centrosomes of dividing cells at all stages of development. During the syncytial cleavage divisions, it is present in the centrosomes. After cellularization, during embryogenesis and imaginal disc development, it is localized to the centrosome only during mitosis, and is redistributed into the cytoplasm during interphase. At mid- oogenesis, it is localized to the minus ends of microtubules in the oocyte. Initial cloning and molecular characterization of the gene encoding centrosomin revealed the presence of three overlapping, alternatively spliced transcripts. The three resulting mRNA encode very similar polypeptides, differing only at their amino termini, and are designated CNN-1, CNN-2 and CNN-3. All three transcripts share 5 exons. Two of the transcripts are found in somatic tissue, while the third is expressed in testes.
Mutations in centrosomin have been isolated that result in male and female sterility. Using these sterile alleles, it has been shown that centrosomin is required for the assembly of functional centrosomes during the syncytial cleavage. During the syncytial cleavage divisions, the loss of functional centrosomin from the centrosomes results in dramatic defects in nuclear division and spindle organization. Aberrant division and nuclear fusion can be seen from the early cycles of nuclear divisions. Mutant embryos do not cellularize. Examination of spindle morphology and centrosomal composition suggest that mutant embryos lack fully functional centrosomes. Most of the spindles in mutant animals are anastral. Chromosomes apparently organize spindles in mutant embryos in the absence of functional centrosomes. These results suggest that maternally supplied centrosomin is required for the assembly of functional centrosomes during cleavage.
SUMMARY OF THE INVENTION
Therefore, according to present invention, there is provided, in a first embodiment, an isolated CNN-4 polypeptide.2. The CNN-4 may be vertebrate, mammalian, Drosophila melanogaster, or human CNN-4. In particular, the CNN-4 has the sequence of SEQ LD NO:2. Also provide are isolated peptides having between about 10 and about 50 consecutive residues of CNN-4, for example, about 15, about 20, about 25, about 30, about 35, about 40, or about 50 residues. The peptide may be about 10 to about 100 residues in length, for example about 10, about 15, about 20, about 25, about 30, about 40, or about 50 residues. The peptide may be conjugated to a carrier molecule, for example, KLH or BSA.
In another embodiment, there is provided a monoclonal antibody that binds immunologically to CNN-4. The monoclonal antibody may be species specific, may further comprises a detectable label, which may be selected from a fluorescent label, a chemilluminescent label, a radiolabel and an enzyme. Also provided is a hybridoma cell that produces a monoclonal antibody that binds immunologically to CNN-4. In a related embodiment, there is provided a polyclonal antisera, antibodies of which bind immunologically to CNN-4.
In yet another embodiment, there is provided an isolated polynucleotide encoding CNN-4, or the complement thereof. The isolated polynucleotide may be derived from vertebrate, mammalian, Drosophila or human sources. The polynucleotide may have the amino acid sequence of SEQ ID NO:2, and may have the polynucleotide sequence of SEQ LD NO: l, or the complement thereof. The polynucleotide may be genomic DNA, complementary DNA and RNA. In a related embodiment, the polynucleotide may be an expression cassette comprising a DNA segment encoding CNN-4 and a promoter operably linked to said segment. The expression cassette may further comprise a polyadenylation signal operably linked to said segment, for example, a tissue specific promoter, an inducible promoter or a constitutive promoter. In another embodiment, the expression cassette is located in a replicable cloning vector which includes an origin of replication. The replicable vector may be a viral vector selected from the group consisting of retrovirus, adenovirus, herpesvirus, vaccinia virus, polyoma virus and adeno- associated virus. The replicable vector may be packaged in a virus particle or in a liposome. The replicable vector is about 5000 bases, about 10,000 bases, about
15,000 bases, about 20,000 bases, about 25,000 bases, about 30,000 bases, about 35,000 bases, about 40,000 bases, about 45,000 bases, about 50,000 bases, about 75,000 bases and about 100,000 bases.
In still yet another embodiment, there is provided an isolated oligonucleotide of between about 10 and about 100 bases, the sequence of which comprises between about 10 and 100 consecutive bases of SEQ ID NO:l. The oligonucleotide may be about 10 bases, about 15 bases, about 20 bases, about 30 bases, about 40 bases, about 50 bases, about 75 bases, about 100 bases in length.
Ln still a further embodiment, there is provided a method for expressing a CNN-4 polypeptide comprising the steps of (i) providing a cell; (ii) introducing into said cell an expression cassette comprising a DNA segment encoding a CNN-4 polypeptide and a promoter operably linked to said segment; and (iii) culturing said cell under conditions permitting expression of CNN-4.
In yet a still further embodiment, there is provided a method for identifying a CNN-4-binding protein comprising the steps of (u) providing a CNN-4 polypeptide; (ii) contacting said CNN-4 polypeptide with a sample; and (iii) identifying a polypeptide bound to said CNN-4 polypeptide. The method may comprise a two- hybrid selection system. In another embodiment, there is provided a method of isolating a CNN-4 related gene comprising the steps of (i) selecting a probe consisting essentially of conserved regions from CNN-4; (ii) hybridizing said probe to RNA or DNA from a candidate cell; and (iii) isolating the hybridizing RNA or DNA. The method may further comprise cloning said hybridizing DNA.
In yet another embodiment, there is provided a method of selecting a cell comprising the steps of (i) transforming a population of cells with an expression cassette comprising a DNA segment encoding a CNN-4 and a promoter operably linked to said segment; (ii) culturing cells under conditions suitable for the expression of CNN-4; and (iii) selecting a cell that survives step (ii). The expression cassette may be part of a larger polynucleotide further comprising a second gene, for example, where the second gene is flanked by genomic sequences of a target site in said cell, and the CNN-4 gene is not flanked by said genomic sequences.
In still yet another embodiment, there is provided a method for selecting a cell comprising (i) transforming a population of cells with an expression cassette comprising a DNA segment encoding CNN-4 and a promoter operably linked to said segment; (ii) culturing cells under conditions suitable for the expression of CNN-4; and (iii) selecting a cell is killed in step (ii). The promoter may be inducible and the conditions result in the induction of said promoter. The method also may comprise a step, between steps (i) and (ii), of making a replicate culture of transformed cells. The expression cassette may be part of a larger polynucleotide further comprising a second gene, for example, where the second gene is a selectable marker. The selectable marker may confer resistance to neomycin, hygromycin, puromycin, zeocin, mycophenolic acid, histidinol or methotrexate.
In still another embodiment, there is provided a method of diagnosing a hyperproliferative disease comprising the steps of (i) obtaining a sample from a subject; and (ii) assaying CNN-4 in cells of said sample. The hyperproliferative disease may be cancer, for example, a cancer of the brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood cancer. The assaying may comprise measuring CNN-4 mRNA levels in said sample, measuring CNN-4 protein levels in said sample, e.g., an immunoassay. The assaying also may comprise characterizing the structure of a CNN-4 polynucleotide in said sample, for example, by sequencing. The method also may further comprise the step of comparing the CNN-4 of said sample with that of a non- diseased sample.
In still yet another embodiment, there is provided a method for altering the hyperproliferative phenotype of a cell comprising the step of contacting the cell with
CNN-4 under conditions permitting the uptake of CNN-4 by said cell. The phenotype may be selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth, metastasis and blocking mitosis. The CNN-4 may be encapsulated in a liposome.
In still a further embodiment, there is provided a method for altering the phenotype of a hyperproliferative cell comprising the step of introducing into said cell an expression cassette comprising (i) a DNA segment encoding CNN-4 and (ii) a promoter active in cell, wherein said promoter is operably linked to said segment, under conditions permitting the uptake of said expression cassette by said hyperproliferative cell. The phenotype may be selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth metastasis and blocking mitosis, and the expression cassette may be encapsulated in a liposome, may be contained in a viral vector selected from the group consisting of retro virus, adenovirus, adeno-associated virus, vaccinia virus and herpesvirus. The viral vector may be packaged in a viral particle.
In still other embodiments, there are provided (i) a method for treating a hyperproliferative disease in a subject comprising the step of introducing into a hyperproliferative cell in said subject a CNN-4 polypeptide, and (ii) a method for treating a hypeφroliferative disease in a subject comprising the step of introducing into a hypeφroliferative cell in said subject an expression cassette comprising (i) a DNA. segment encoding CNN-4 and (ii) a promoter active in cell, wherein said promoter is operably linked to said segment, under conditions permitting the uptake of said nucleic acid by said tumor cell.
In other aspects of the invention, there are provided a non-human transgenic organisms, the cells of which comprise a transgenic CNN-4 gene. The transgenic CNN-4 gene may be heterologous or homologous to said organism, may be under the control of a promoter (tissue specific, inducible). The CNN-4 may be a mutant CNN- 4 gene; both endogenous copies of CNN-4 may be dysfunctional; the organism is a vertebrate, mammal, Drosophila, or mouse.
In yet another aspect, the invention provides, a method of screening a candidate substance for proliferative activity comprising the steps of (i) providing a cell lacking functional CNN-4; (ii) contacting said cell with said candidate substance; and (iii) determining the effect of said candidate substance on said cell. The determining may comprise comparing one or more characteristics of the cell in the presence of said candidate substance with characteristics of a cell in the absence of said candidate substance, for example, CNN-4 expression, proliferation, metastasis, contact inhibition, soft agar growth, blocking mitosis, tumor formation, tumor progression and tissue invasion. The cell may be contacted in vitro or in vivo.
In yet further aspects, the present invention provides a method for increasing proliferation of cellular population comprising the step of inhibiting CNN-4 function in at least one cell of said population. The inhibition may be achieved by introducing into a cell of said population an expression cassette comprising (i) a DNA segment encoding at least a portion of a CNN-4 complementary sequence and (ii) a promoter active in cell, wherein said promoter is operably linked to said DNA segment, under conditions permitting the uptake of said nucleic acid by said tumor cell. The DNA segment may be complementary to an exon of CNN-4, an intron of CNN-4, adjacent intronic and exonic regions of CNN-4, a splice junction of CNN-4 or a translation initiation site of CNN-4. Alternatively, the inhibition may be achieved by introducing into a cell of said population an expression cassette comprising (i) a DNA segment encoding a single-chain antibody that binds immunologically to CNN-4 and (ii) a promoter active in cell, wherein said promoter is operably linked to said DNA segment, under conditions permitting the uptake of said nucleic acid by said tumor cell.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIG. 1 - Map of the Centrosomin Transcription Units. Splicing patterns for centrosomin (cnn) transcripts: CNN-1 (top); CNN-2 (next to top); CNN-3 (next to bottom); CNN-4 (bottom). Also shown are genomic regions with restriction enzyme sites (E - EcoRl; H - HinΩTΑ, S - Sail).
FIG. 2 - Map of the Centrosomin Genes, mRNAs and Polypeptides.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
Though considerable research has been conducted on how the normal cell cycle operates, and how it can be disrupted, there remain many questions about this process. One of the key questions yet to be answered is precisely which molecules are responsible for the carefully choreographed series of steps which result in cell division. The answer has far reaching implications in many aspects of biology, including developmental biology and hypeφroliferative diseases.
I. The Present Invention The present invention arises, in one aspect, from the discovery of a new gene, now designated as CNN-4. This gene is part of a complex transcription unit previously identified in connection with three closely related centrosomin transcripts. FIG. 1. The CNN-4 gene shares three exons with CNN-1, CNN-2 and CNN-3, but includes three unique exons that are spliced out of the centrosomin transcripts. The resulting polypeptide contains regions corresponding to exons lc, 3, 4, 4a, 4b and 4c. FIG. 2.
It has been determined that CNN-4 plays an important role in cell cycle regulation. More particularly, CNN-4 is capable of blocking cell division. Based on its lack of homology with other cell cycle proteins, it is believed that the mechanism by which this occurs is previously unknown. Because of this function, CNN-4, and related compositions, will find use in a variety of different contexts. For example, it may be useful to block CNN-4 activity in cells in culture in order to increase their viability and longevity. It also may prove useful to block CNN-4 activity in order to increase proliferation of tissues in vivo, for example, in tissues that have been damaged and cannot normally repair themselves by increases in number due to cell division. It also is contemplated that increasing CNN-4 activity will find use in treating various hypeφroliferative diseases, such as cancer. Finally, defects in CNN-4 may lead to hypeφroliferative disorders and, hence, determining the status of the CNN-4 gene product provides a diagnostic tool for such disease states.
The embodiments, as well as others, are set forth in the following detailed description of the invention.
II. Peptides and Polypeptides
In one embodiment of the present invention, there is provided a cell cycle regulator designated CNN-4. This molecule has been shown to block cell division. In addition to the entire CNN-4 molecule, the present invention also relates to fragments of the polypeptide that may or may not retain the ability to block cell division. Fragments, including varying portions of the N-terminus (truncations) of the molecule may be generated by genetic engineering of translation stop sites within the coding region (discussed below). Alternatively, treatment of the CNN-4 molecule with proteolytic enzymes, known as proteases, can produce a variety of N-terminal, C- terminal and internal fragments. Synthetic production of short peptides also is contemplated. Examples of fragments may include contiguous residues of the CNN-4 sequence given in SEQ ID NO:2 of 6, 7, 8, 9, 10, 11, 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. Structural Features of the Polypeptide The gene for CNN-4 encodes a 462 amino acid polypeptide. The predicted molecular weight of this molecule is 52259.38 with a resulting pi of 7.50. Thus, at a minimum, this molecule may be used as a standard in assays where molecule weight and pi are being examined.
Sequence analysis of CNN-4 indicates that it is principally coiled coil in nature. The amino and carboxy ends as well as two smaller regions in the interior of the protein have low helical potential. However the central domain from amino acid residues 40 to 69, 80 to 126 and 152 to 342 all have robust predictions for forming alpha-helical coils. Included in this general coil structure is the prdiction of a leucine zipper extending from residue 45 to 66 within the first coiled domain mentioned above. The protein also has consensus sites for phosphorylation, myrystilization and glycosylation. The number and postion of these sites is given below. BLAST serches of the genome databases reveal no striking homology to any known proteins. Weak homology is found for the coiled coil domains proteins such as myosin. The coiled coil character of CNN-4 and the presence of a leucine zipper indicates that this protein likely dimerizes with itself and/or forms heteromeric complexes with other proteins. The nature of these other proteins is at present unknown but is the topic of current investigation.
Table 1 - Putative Phosphorylation Sites
CK2 Sites PKC Sites Myristylation Sites Glvcosylation Sites (N-linked) 41-44 3-5 69-74 4-7 77-80 41-43 361-366 19-22
115-118 95-97 363-368 232-235
201-204 158-160 372-377 380-383
230-233 237-239 444-447
269-272 255-257
366-369 269-271 290-292 321-323 395-397
B. Functional Aspects
When the present application refers to the function of CNN-4 or "wild-type" activity, it is meant that the molecule in question has the ability to block cell division of a cell. Other phenotypes that may be considered to be regulated by CNN-4 are angiogenesis, adhesion, migration, cell-to-cell signaling, cell growth, cell proliferation, density-dependent growth, anchorage-dependent growth, tumor formation, metastasis and others. Determination of which molecules possess this activity may be achieved using assays familiar to those of skill in the art.
As stated above, the identification of a leucine zipper motif suggests possible dimerization or multimerization with itself (homodimers or homomultimers) or other molecules (heterodimers or heteromultimers).
C. Variants of CNN-4 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 function or immunogenic activity, and are exemplified by variants lacking the noncoiled sequences 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 of the 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.
The following is a discussion based upon changing of the amino acids of a protein to create an equivalent, or even an improved, second-generation molecule. 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 DNA sequences of genes without appreciable loss of their biological utility or activity, as discussed below. Table 2, below, 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 of the resultant protein, which in turn defines the interaction of the 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, i.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, incoφorated 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 imetics. 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 of the natural properties of CNN-4, but with altered and even improved characteristics.
D. 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. One particular fusion of interest would include a deletion construct lacking one or more of the phosphorylation sites of CNN-4 or the leucine zipper motif. Fusion to a polypeptide that can be used for purification of the substrate-CNN-4 complex would serve to isolated the substrate for identification and analysis.
E. Purification of Proteins
It will be desirable to purify CNN-4, or 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 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 even 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 of the composition, such as constituting about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or more of the proteins in the composition.
Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure.
These include, for example, determining the specific activity of an active fraction, or assessing the 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 activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "-fold purification number."
The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.
Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.
There is no general requirement that the protein or peptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different forms of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an
HPLC apparatus will generally result in a greater "-fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.
It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.
High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain 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 of the sample.
Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with tiny particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the sole factor determining rate of flow is the size. Hence, molecules are eluted from the column in decreasing size, so long as the shape is relatively constant. Gel chromatography is unsuφassed for separating molecules of different size because separation is independent of all other factors such as pH, ionic strength, temperature, etc. There also is virtually no adsoφtion, less zone spreading and the elution volume is related in a simple matter to molecular weight.
Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule that it can specifically bind to. This is a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material is then able to specifically adsorb the substance from the solution. Elution occurs by changing the conditions to those in which binding will not occur (alter pH, ionic strength, temperature, etc.).
A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.
The matrix should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The ligand should be coupled in such a way as to not affect its binding properties. The ligand should also provide relatively tight binding. And it should be possible to elute the substance without destroying the sample or the ligand. One of the most common forms of affinity chromatography is immunoaffinity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed below. F. Synthetic Peptides
The present invention also describes smaller CNN-4-related peptides for use in various embodiments of the present invention. Because of their relatively small size, the peptides of the 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 incoφorated herein by reference. Short peptide sequences, or libraries of overlapping peptides, usually from about 6 up to about 35 to 50 amino acids, which correspond to the selected regions described herein, can be readily 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 of the invention is inserted into an expression vector, transformed or transfected into an appropriate host cell and cultivated under conditions suitable for expression.
G. Antigen Compositions
The present invention also provides for the use of CNN-4 proteins or peptides as antigens for the immunization of animals relating to the production of antibodies. It is envisioned that either CNN-4, 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 are the carriers are keyhole limpet hemocyannin (KLH) or bovine serum albumin (BSA).
III. Oliogo- and Polynucleotides
The present invention also provides, in another embodiment, oligo- and polynucleotides genes relating to CNN-4. The gene for Drosophila CNN-4 has been identified, as well as variants thereof. However, the present invention is not limited in scope to these genes, however, as one of ordinary skill could, using such molecules, and related tools, identify related homologs in various other species (e.g., mouse, rat, rabbit, monkey, gibbon, chimp, ape, baboon, human cow, pig, horse, sheep, dog, cat and other species).
In addition, it should be clear that the present invention is not limited to the specific polynucleotides disclosed herein. As discussed below, a "CNN-4 gene" may contain a variety of different bases and yet still produce a corresponding polypeptide that is functionally indistinguishable, and in some cases structurally, from the fruit fly genes disclosed herein.
Similarly, any reference to a oligo- or polynucleotide should be read as encompassing a host cell containing that molecule and, in some cases, capable of expressing the a related protein product. In addition to therapeutic considerations, cells expressing oligo- and polynucleotide 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 CNN-4.
A. Polynucleotides Encoding CNN-4 The Drosophila sequences disclosed in SEQ LD NOS: l and 3 are polynucleotides according to the present invention. Polynucleotides, or DNA or RNA segments according to the present invention may encode an entire CNN-4 gene, including or excluding introns, an exon of CNN-4, a domain of CNN-4 that blocks cell division, or any other fragment of the CNN-4 sequences set forth herein. The polynucleotide 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 CNN-4 gene is made up of 6 exons, designated as lc, 3, 4, 4a, 4b and 4c. The lc, 3 and 4 exons are shared with the centrosomin transcripts CNN-1 to -3, but exons 4a-c are not. Rather, these exons arise from an intronic region that is completely spliced out of the other centrosomin transcripts. The entire intron is 1296 bases in length and spliced 4a-c segment is 1009 in length.
The term "cDNA" is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The entire spliced coding region of CNN-4 is 1812 nucleotides, and the entire cDNA is 1832 nucleotides. 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.
The six exons of CNN-4 are located at the following positions in SEQ LD NO: 1 :
exon lc = 15-176 exon 4a = 818-986 exon 3 = 177-550 exon 4b = 987-1338 exon 4 = 551-818 exon 4c = 1339-1826
The genomic sequence from the entire centrosomin region is illustrated by SEQ LD NO:3. The exonic positions are as follows:
exon la = 194-502 exon 4a = 6281-6449 exon lb = 3346-3525 exon 4b = 6518-6869 exon lc (CNN-3) = 5252-5413 exon 4c = 6930-7417 exon lc (CNN-4) = 5268-5413 exon 5 = 7493-7687 exon 2 = 5034-5077 exon 6 = 8105-10229 exon 3 = 5486-5859 exon 7 = 10293-11302 exon 4 = 5930-6196
It also is contemplated that a given CNN-4 gene from a given species may be represented by natural variants that have slightly different nucleic acid sequences but, nonetheless, encode the same protein. Table 2. As used in this application, the term "an isolated polynucleotide" refers to a polynucleotide molecule that has been isolated, to some extent, from its natural state, i.e., away from 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 2, below), and also refers to codons that encode biologically equivalent amino acids, as discussed in the following pages.
TABLE 2
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 cue 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 Tφ w UGG
Tyrosine Tyr Y UAC UAU
Allowing for the degeneracy of the genetic code, sequences that have at least about 50%, 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 SEQ LD NO: 1 are encompassed in by the present invention. Sequences that are essentially the same as those set forth in SEQ LD NO:l also may be functionally defined as sequences that are capable of hybridizing to a polynucleotide segment containing the complement of SEQ LD NO:l under standard conditions.
The DNA segments of the present invention include those encoding biologically functional equivalent CNN-4 proteins and peptides, as described above. Such sequences may arise as a consequence of codon redundancy and amino acid functional equivalency that are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, 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. 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 the sequence set forth in SEQ ID NO:l. 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 the nucleic acid segment of SEQ LD NO: 1 under relatively stringent conditions such as those described herein. Such sequences may encode the entire CNN-4 protein or functional or non-functional fragments thereof.
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, 11, 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, 1386, 1500, 1812, 1832, 2000, 2500, 3000 or 4000, 5000, 10,000, 12,580 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 KC1, 3 mM MgCl2, 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 KC1,
1.5 μM MgCl , 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 CNN-4 or, more particularly, homologs of CNN-4 from other 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, incoφorating 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 of the 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.
Ln 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 of the 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 hydroxyl amine, to obtain sequence variants.
C. Antisense Constructs
In some cases, it may prove useful to abrogate the function of CNN-4 in normal cells, or limit the proliferative effects of a mutant CNN-4. Antisense treatments are one way of addressing this situation. Antisense technology also may be used to "knock-out" function of CNN-4 in the development of cell lines or transgenic mice for research, diagnostic and screening puφoses.
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 eliminating CNN-4 activity is through the use of ribozymes. Although proteins traditionally have been used for catalysis of polynucleotide acids, another class of macromolecules 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") of the 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 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 HLV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that is cleaved by a specific ribozyme. D. Vectors for Cloning, Gene Transfer and Expression
Within certain embodiments expression vectors are employed to express the CNN-4 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 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 of the 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 of the 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 of the 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 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the 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, beta actin 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 puφose.
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.
Where a cDNA insert is employed, one will typically 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, a cell may be identified and selected in vitro or in vivo by including a marker in the expression construct. Such markers 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 heφes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Lmmunologic 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 LRES In certain embodiments of the invention, the use of internal ribosome binding sites (LRES) 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). LRES elements from two members of the picornavirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an LRES from a mammalian message (Macejak and Sarnow, 1991). LRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an LRES, creating polycistronic messages. By virtue of the LRES 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 LRES 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 a polynucleotide that has been cloned therein. In this context, expression does not require that a polypeptide be synthesized.
Knowledge of the 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 (E1A and E1B) encodes proteins responsible for the regulation of transcription of the viral genome and a few cellular genes. The expression of the E2 region (E2A and E2B) results in the synthesis of the proteins for viral DNA replication. These proteins are involved in DNA replication, late gene expression and host cell shut-off (Renan, 1990). The products of the late genes, including the majority of the viral capsid proteins, are expressed only after significant processing of a single primary transcript issued by the major late promoter (MLP). The MLP, (located at 16.8 m.u.) is particularly efficient during the late phase of infection, and all the mRNA's issued from this promoter possess a 5 '-tripartite leader (TPL) sequence which makes them preferred mRNA's for translation.
In a current system, recombinant adenovirus is generated from homologous recombination between shuttle vector and 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 E3 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.
Helper cell lines may be derived from human cells such as human embryonic kidney cells, muscle cells, hematopoietic cells or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from the cells of other mammalian species that are permissive for human adenovirus. Such cells include, e.g., Vero cells or other monkey embryonic mesenchymal or epithelial cells. As stated above, the preferred helper cell line is 293.
Racher et al. (1995) disclosed improved methods for culturing 293 cells and propagating adenovirus. 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 of the adenovirus vector is not believed to be crucial to the successful practice of the invention. The adenovirus may be of any of the 42 different known serotypes or subgroups A-F. Adenovirus type 5 of subgroup C is the preferred starting material in order to obtain the conditional replication- defective adenovirus vector for use in the present invention. This is because Adenovirus type 5 is a human adenovirus about which a great deal of biochemical and genetic information is known, and it has historically been used for most constructions employing adenovirus as a vector.
As stated above, the typical vector according to the present invention is replication defective and will not have an adenovirus 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 of the 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 -10 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 polynucleotide 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 LI 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).
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 heφesviruses 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).
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 DNA segment 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 of the 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, 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 polynucleotide 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 of the 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 have been used for receptor-mediated gene transfer. The most extensively characterized 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 of the 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.
Primary mammalian cell cultures may be prepared in various ways. In order for the cells to be kept viable while in vitro and in contact with the expression construct, it is necessary to ensure that the cells maintain contact with the correct ratio of oxygen and carbon dioxide and nutrients but are protected from microbial contamination. Cell culture techniques are well documented and are disclosed herein by reference (Freshner, 1992).
One embodiment of the foregoing involves the blocking of CNN-4 expression to immortalize cells, for example, in the production of a selected polypeptide. Examples of useful mammalian host cell lines are Vero and HeLa cells and cell lines of Chinese hamster ovary, W138, BHK, COS-7, 293, HepG2, NLH3T3, RLN and
MDCK cells. In addition, a host cell strain may be chosen that modulates the expression of inserted sequences, or modifies and process the selected product in the manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for the post- translational processing and modification of proteins. Appropriate cell lines or host systems can be chosen to insure the correct modification and processing of the foreign protein expressed.
Animal cells can be propagated in vitro in two modes: as non-anchorage dependent cells growing in suspension throughout the bulk of the culture or as anchorage-dependent cells requiring attachment to a solid substrate for their propagation (i.e., a monolayer type of cell growth).
Non-anchorage dependent or suspension cultures from continuous established cell lines are the most widely used means of large scale production of cells and cell products. However, suspension cultured cells have limitations, such as tumorigenic potential and lower protein production than adherent T-cells.
Large scale suspension culture of mammalian cells in stirred tanks is a common method for production of recombinant proteins. Two suspension culture reactor designs are in wide use - the stirred reactor and the airlift reactor. The stirred design has successfully been used on an 8000 liter capacity for the production of interferon. Cells are grown in a stainless steel tank with a height-to-diameter ratio of 1: 1 to 3:1. The culture is usually mixed with one or more agitators, based on bladed disks or marine propeller patterns. Agitator systems offering less shear forces than blades have been described. Agitation may be driven either directly or indirectly by magnetically coupled drives. Indirect drives reduce the risk of microbial contamination through seals on stirrer shafts.
The airlift reactor, also initially described for microbial fermentation and later adapted for mammalian culture, relies on a gas stream to both mix and oxygenate the culture. The gas stream enters a riser section of the reactor and drives circulation.
Gas disengages at the culture surface, causing denser liquid free of gas bubbles to travel downward in the downcomer section of the reactor. The main advantage of this design is the simplicity and lack of need for mechanical mixing. Typically, the height-to-diameter ratio is 10: 1. The airlift reactor scales up relatively easily, has good mass transfer of gases and generates relatively low shear forces.
The antibodies of the present invention are particularly useful for the isolation of antigens by immunoprecipitation. Immunoprecipitation involves the separation of the target antigen component from a complex mixture, and is used to discriminate or isolate minute amounts of protein. For the isolation of membrane proteins cells must be solubilized into detergent micelles. Nonionic salts are preferred, since other agents such as bile salts, precipitate at acid pH or in the presence of bivalent cations. Antibodies are and their uses are discussed further, below.
IV. Antibodies In another aspect, the present invention contemplates an antibody that is immunoreactive with a CNN-4 polypeptide 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., Harlowe 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.
Three polyclonal antisera have been raised to portions of CNN-4. All three were raised against peptides that were synthesized in E. coli and purified using a 6X His tag and Ni column chromatography. The first peptide corresponds to the amino acids encoded by exons la, 3, 4, 5 and 6. The peptide was used to immunize guinea pigs. Recovered sera recognizes, in Western blot format, CNN-4 synthesized both in vitro and in vivo. This sera also recognizes CNN-1, -2 and 3 by virtue of the fact that exons 5 and 6 encode amino acids found in these proteins. A second 6X His-tagged peptide, encoded by exons 4a, 4b and 4c, has been synthesized in E. coli, purified as above, and used to immunize both guinea pigs and rats. Antisera from both has been obtained that specifically recognizes CNN-4 from both in vitro and in vivo sources. This sera also has been used to determine the distribution of CNN-4 in immunohistochemically stained preparations.
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 of the 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 ΕLISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to CNN-4-related antigen epitopes. Additionally, it is proposed that monoclonal antibodies specific to the particular CNN-4 of different species may be utilized in other useful applications
In general, both polyclonal and monoclonal antibodies against CNN-4 may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes related to CNN-4. They also may be used in inhibition studies to analyze the effects of CNN-4 related peptides in cells or animals. CNN-4 antibodies will also be useful in immunolocalization studies to analyze the distribution of CNN-4 during various cellular events, for example, to determine the cellular or tissue-specific distribution of CNN-4 polypeptides under different points in the cell cycle. A particularly useful application of such antibodies is in purifying native or recombinant CNN-4, 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 of the present disclosure.
Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988; incoφorated 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, m-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 or partially purified CNN-4 protein, polypeptide or peptide or cell expressing high levels of CNN-4. 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 107 to 2 x 108 lymphocytes.
The antibody-producing B lymphocytes from the immunized animal are then fused with cells of an immortal myeloma cell, generally one of the same species as the animal that was immunized. Myeloma cell lines suited for use in hybridoma-producing fusion procedures preferably are non-antibody-producing, have high fusion efficiency, and enzyme deficiencies that render then incapable of growing in certain selective media which support the growth of only the desired fused cells (hybridomas).
Any one of a number of myeloma cells may be used, as are known to those of skill in the art (Goding, 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, MPC11-X45-GTG 1.7 and S194/5XX0 Bui; for rats, one may use R210.RCY3, Y3-Ag 1.2.3, LR983F 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 10" to 1 x 10" . 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 supematants (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. V. Methods for Screening of Active Compounds
The present invention also contemplates the use of CNN-4 and active fragments, and polynucleotides coding therefor, in the screening of compounds for activity in either stimulating CNN-4 activity, overcoming the lack of CNN-4, blocking the effect of CNN-4, or overcoming the effects of a mutant CNN-4. These assays may make use of a variety of different formats and may depend on the kind of "activity" for which the screen is being conducted. Contemplated functional "read-outs" include binding to a compound, inhibition of binding to a substrate, ligand, receptor or other binding partner by a compound, inhibition or stimulation of cell-to-cell signaling, growth, metastasis, cell division, cell migration, soft agar colony formation, contact inhibition, tissue invasiveness, angiogenesis, apoptosis, tumor establishment or progression or other malignant phenotype.
As used herein the term "candidate substance" may be a protein or fragment thereof, a small molecule inhibitor, an oligo- or polynucleotide, of natural or synthetic origin. It may prove to be the case that the most useful pharmacological compounds for identification through application of the screening assay will be compounds that are structurally related to CNN-4 or targets of CNN-4. The active compounds may include fragments or parts of naturally-occurring compounds or may be only found as active combinations of known compounds which are otherwise inactive.
Accordingly, the active compounds may include fragments or parts of naturally-occurring compounds or may be found as active combinations of known compounds which are otherwise inactive. Accordingly, the present invention provides screening assays to identify agents from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be polypeptide or fragment thereof, oligonucleotide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design. "Effective amounts" in certain circumstances are those amounts effective to reproducibly produce the assayed result.
A. In Vitro Assays
In one embodiment, the invention is to be applied for the screening of compounds that bind to the CNN-4 polypeptide or fragment thereof. The polypeptide or fragment may be either free in solution, fixed to a support, expressed in or on the surface of a cell. Either the polypeptide or the compound may be labeled, thereby permitting determining of binding.
In another embodiment, the assay may measure the inhibition of binding of CNN-4 to a natural or artificial substrate or binding partner. Competitive binding assays can be performed in which one of the agents (CNN-4, binding partner or compound) is labeled. Usually, the polypeptide will be the labeled species. One may measure the amount of free label versus bound label to determine binding or inhibition of binding.
Another technique for high throughput screening of compounds is described in WO 84/03564. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. The peptide test compounds are reacted with CNN-4 and washed. Bound polypeptide is detected by various methods.
Purified CNN-4 can be coated directly onto plates for use in the aforementioned drug screening techniques. However, non-neutralizing antibodies to the polypeptide can be used to immobilize the polypeptide to a solid phase. Also, fusion proteins containing a reactive region (preferably a terminal region) may be used to link the CNN-4 active region to a solid phase.
Various cell lines containing wild-type CNN-4, or natural or engineered mutations in CNN-4, can be used to study various functional attributes of CNN-4 and how a candidate compound affects these attributes. Methods for engineering mutations are described elsewhere in this document. In such assays, the compound would be formulated appropriately, given its biochemical nature, and contacted with a target cell. Depending on the assay, culture may be required. The cell may then be examined by virtue of a number of different physiologic assays. Alternatively, molecular analysis may be performed in which the function of CNN-4, or related pathways, may be explored. This may involve assays such as those for protein expression, protein function, substrate utilization, etc.
B. In Vivo Assays
The present invention also encompasses the use of various whole organism models. By developing or isolating mutant cells lines that fail to express normal CNN-4, one can generate cancer models in mice that will be highly predictive of cancers in humans and other mammals. These models may employ the orthotopic or systemic administration of tumor cells to mimic primary and or metastatic cancers.
Alternatively, one may induce cancers in animals by providing agents known to be responsible for certain events associated with malignant transformation and/or tumor progression. Finally, transgenic animals (discussed below) that lack a wild-type CNN-4 may be utilized as models for cancer development and treatment.
Treatment of animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal. Administration will be by any route the could be utilized for clinical or non-clinical puφoses, including but not limited to oral, nasal, buccal, rectal, vaginal or topical. Alternatively, administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Specifically contemplated are systemic intravenous injection, regional administration via blood or lymph supply and intratumoral injection.
Determining the effectiveness of a compound in vivo may involve a variety of different criteria. Such criteria include, but are not limited to, survival, reduction of tumor burden or mass, arrest or slowing of tumor progression, elimination of tumors, inhibition or prevention of metastasis, increased activity level, improvement in immune effector function and improved food intake.
C. Rational Drug Design The goal of rational drug design is to produce structural analogs of biologically active polypeptides or compounds with which they interact (agonists, antagonists, inhibitors, binding partners, etc.). By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for
CNN-4 or a fragment thereof. This could be accomplished by x-ray crystallography, computer modeling or by a combination of both approaches. An alternative approach, "alanine scan," involves the random replacement of residues throughout molecule with alanine, and the resulting affect on function determined.
It also is possible to isolate a CNN-4-specific antibody, selected by a functional assay, and then solve its crystal structure. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.
Thus, one may design molecules which have improved CNN-4 activity or which act as stimulators, inhibitors, agonists, antagonists of CNN-4 or molecules affected by CNN-4 function. By virtue of the availability of cloned CNN-4 sequences, sufficient amounts of CNN-4 can be produced to perform crystallographic studies. In addition, knowledge of the polypeptide sequences permits computer employed predictions of structure-function relationships. VI. Methods for Immortalizing Cell Lines
According to the present invention, it may prove desirable to antagonize the anti-proliferative effects of CNN-4, thereby resulting in increased cellular proliferation and, in some instances immortalization of the cell. The ability to immortalize certain cell types can have a significant benefit. For example, manipulation of certain cells that are hard to maintain in culture over long periods of time (lymphocytes) and cells that can be immortalized, but lose their primary cell characteristics (pancreatic β-cells), may be improved by blocking CNN-4 function.
Generally, four methods for decreasing CNN-4 function are available. The first method involves the use of a pharmaceuticals. The second method involves the use of an antisense construct designed to inhibit CNN-4 transcription, splicing, or translation. This aspect is described in greater detail above. The third method involves a CNN-4 directed ribozyme, also as described above. The fourth method involves use of a genetic construct encoding for a single chain antibody directed to CNN-4. This embodiment is discussed further, below.
Methods for the production of single-chain antibodies are well known to those of skill in the art. The skilled artisan is referred to U.S. Patent 5,359,046,
(incoφorated herein by reference) for such methods. A single-chain antibody is created by fusing together the variable domains of the heavy and light chains using a short peptide linker, thereby reconstituting an antigen binding site on a single molecule. Single-chain antibody variable fragments (scFvs) in which the C-terminus of one variable domain is tethered to the N-terminus of the other via a 15 to 25 amino acid peptide or linker, have been developed without significantly disrupting antigen binding or specificity of the binding (Bedzyk et al., 1990; Chaudhary et al., 1990). These Fvs lack the constant regions (Fc) present in the heavy and light chains of the native antibody.
Single-chain antibodies can be synthesized in a host cell, targeted to particular cellular compartments, and used to interfere, in a highly specific manner, with cell growth and metabolism (Richardson and Marasco, 1995). Recently, single-chain antibodies were utilized for the phenotypic knockout of growth-factor receptors, the functional inactivation of p21rαs, and the inhibition of HIV-1 replication. Intracellular antibodies offer a simple and effective alternative to other forms of gene inactivation, as well as demonstrate a clear potential as reagents for cancer therapy and for the control of infectious diseases. Single-chain antigen-binding proteins also represent potentially unique molecules for targeted delivery of drugs, toxins, or radionuclides to a tumor site, and show increased accessibility to tumor cells in vivo (Yokoda et al, 1992).
VII. Methods for Diagnosing Hyperproliferative Disorders
Given CNN-4' s involvement with cell cycle regulation, it is proposed that some hypeφroliferative diseases may be caused by aberrations in this gene. Therefore, CNN-4 and the corresponding gene may be targeted in diagnostic or prognostic indications of such disease. More specifically, point mutations, deletions, insertions or regulatory pertubations relating to CNN-4 may cause or promote hypeφroliferative diseases such as cancers. Other phenomena associated with malignancy that may be affected by CNN-4 expression include metastasis, angiogenesis and tissue invasion.
A. Genetic Diagnosis
One embodiment of the instant invention comprises a method for detecting variation in the expression of CNN-4. This may comprises determining that level of CNN-4 or determining specific alterations in the expressed product. This sort of assay has importance in the diagnosis of hypeφroliferative diseases, including cancers.
Such cancers may involve cancers of the brain, lung, liver, spleen, kidney, pancreas, small intestine, blood cells, lymph node, colon, breast, endometrium, stomach, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue.
The 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.
Polynucleotides are 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 polynucleotide of interest is identified in the sample directly using amplification or with a second, known oligo- or polynucleotide 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 chemilluminescence, radioactive scintigraphy of radiolabel or fluorescent label or even via a system using electrical or thermal impulse signals (Affymax Technology; 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 CNN-4-related pathologies. In this way, it is possible to correlate the amount or kind of CNN-4 detected with various clinical states.
A cell takes a genetic step toward oncogenic transformation when one allele of a cell cycle regulatory gene is inactivated due to inheritance of a germline lesion or acquisition of a somatic mutation. The inactivation of the other allele of the gene usually involves a somatic micromutation or chromosomal allelic deletion that results in loss of heterozygosity (LOH). Alternatively, both copies of a gene may be lost by homozygous deletion.
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 preferred. 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 (32P, 14C, 35S, 3H, or other label), with a fluorophore (rhodamine, fluorescein) or a chemillumiscent (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. Patents 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 primers 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 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. 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/US 89/01025, 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 (incoφorated 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 transcription of many RNA copies of the sequence. Other amplification methods include "RACE" and "one-sided PCR™" (Frohman, M.A., In: RCR™ 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 a polynucleotide 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), incoφorated 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 puφose 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 of the 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 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 the CNN-4 gene that may then be analyzed by direct sequencing.
(vi) Kit Components All the essential materials and reagents required for detecting and sequencing
CNN-4 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, in suitable means, distinct containers for each individual reagent and enzyme as well as for each primer or probe. (vii) Design and Theoretical Considerations for Relative Quantitative
RT-PCR™
Reverse transcription (RT) of RNA to cDNA followed by relative quantitative PCR™ (RT-PCR™) can be used to determine the relative concentrations of specific mRNA species isolated from patients. By determining that the concentration of a specific mRNA species varies, it is shown that the gene encoding the specific mRNA species is differentially expressed.
In PCR , the number of molecules of the amplified target DNA increase by a factor approaching two with every cycle of the reaction until some reagent becomes limiting. Thereafter, the rate of amplification becomes increasingly diminished until there is no increase in the amplified target between cycles. If a graph is plotted in which the cycle number is on the X axis and the log of the concentration of the amplified target DNA is on the Y axis, a curved line of characteristic shape is formed by connecting the plotted points. Beginning with the first cycle, the slope of the line is positive and constant. This is said to be the linear portion of the curve. After a reagent becomes limiting, the slope of the line begins to decrease and eventually becomes zero. At this point the concentration of the amplified target DNA becomes asymptotic to some fixed value. This is said to be the plateau portion of the curve.
The concentration of the target DNA in the linear portion of the PCR amplification is directly proportional to the starting concentration of the target before the reaction began. By determining the concentration of the amplified products of the target DNA in PCR reactions that have completed the same number of cycles and are in their linear ranges, it is possible to determine the relative concentrations of the specific target sequence in the original DNA mixture. If the DNA mixtures are cDNAs synthesized from RNAs isolated from different tissues or cells, the relative abundances of the specific mRNA from which the target sequence was derived can be determined for the respective tissues or cells. This direct proportionality between the concentration of the PCR products and the relative mRNA abundances is only true in the linear range of the PCR reaction. The final concentration of the target DNA in the plateau portion of the curve is determined by the availability of reagents in the reaction mix and is independent of the original concentration of target DNA. Therefore, the first condition that must be met before the relative abundances of a mRNA species can be determined by RT-PCR for a collection of RNA populations is that the concentrations of the amplified PCR products must be sampled when the PCR™ reactions are in the linear portion of their curves.
The second condition that must be met for an RT-PCR™ experiment to successfully determine the relative abundances of a particular mRNA species is that relative concentrations of the amplifiable cDNAs must be normalized to some independent standard. The goal of an RT-PCR experiment is to determine the abundance of a particular mRNA species relative to the average abundance of all mRNA species in the sample. In the experiments described below, mRNAs for β- actin, asparagine synthetase and lipocortin π were used as external and internal standards to which the relative abundance of other mRNAs are compared.
Most protocols for competitive PCR utilize internal PCR standards that are approximately as abundant as the target. These strategies are effective if the products of the PCR™ amplifications are sampled during their linear phases. If the products are sampled when the reactions are approaching the plateau phase, then the less abundant product becomes relatively over represented. Comparisons of relative abundances made for many different RNA samples, such as is the case when examining RNA samples for differential expression, become distorted in such a way as to make differences in relative abundances of RNAs appear less than they actually are. This is not a significant problem if the internal standard is much more abundant than the target. If the internal standard is more abundant than the target, then direct linear comparisons can be made between RNA samples. The above discussion describes theoretical considerations for an RT-PCR assay for clinically derived materials. The problems inherent in clinical samples are that they are of variable quantity (making normalization problematic), and that they are of variable quality (necessitating the co-amplification of a reliable internal control, preferably of larger size than the target). Both of these problems are overcome if the
RT-PCR is performed as a relative quantitative RT-PCR with an internal standard in which the internal standard is an amplifiable cDNA fragment that is larger than the target cDNA fragment and in which the abundance of the mRNA encoding the internal standard is roughly 5-100 fold higher than the mRNA encoding the target. This assay measures relative abundance, not absolute abundance of the respective mRNA species.
Other studies may be performed using a more conventional relative quantitative RT-PCR™ assay with an external standard protocol. These assays sample the PCR products in the linear portion of their amplification curves. The number of PCR cycles that are optimal for sampling must be empirically determined for each target cDNA fragment. In addition, the reverse transcriptase products of each RNA population isolated from the various tissue samples must be carefully normalized for equal concentrations of amplifiable cDNAs. This consideration is very important since the assay measures absolute mRNA abundance. Absolute mRNA abundance can be used as a measure of differential gene expression only in normalized samples. While empirical determination of the linear range of the amplification curve and normalization of cDNA preparations are tedious and time consuming processes, the resulting RT-PCR assays can be superior to those derived from the relative quantitative RT-PCR assay with an internal standard.
One reason for this advantage is that without the internal standard/competitor, all of the reagents can be converted into a single PCR product in the linear range of the amplification curve, thus increasing the sensitivity of the assay. Another reason is that with only one PCR product, display of the product on an electrophoretic gel or another display method becomes less complex, has less background and is easier to inteφret.
(viii) Chip Technologies Specifically contemplated by the present inventors are chip-based DNA technologies such as those described by Hacia et al (1996) and Shoemaker et al. (1996). Briefly, these techniques involve quantitative methods for analyzing large numbers of genes rapidly and accurately. By tagging genes with oligonucleotides or using fixed probe arrays, one can employ chip technology to segregate target molecules as high density arrays and screen these molecules on the basis of hybridization. See also Pease et al. (1994); Fodor et al. (1991).
B. Immunodiagnosis
Antibodies of the present invention can be used in characterizing the CNN-4 content of healthy and diseased tissues, through techniques such as ELISAs 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-CNN-4 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 adsoφtion 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 immunoaffinity for CNN-4 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 H2O2, 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.
VIII. Methods for Treating Hyperproliferative Disorders
The present invention also involves, in another embodiment, the treatment of hypeφroliferative diseases. Virtually any hypeφroliferative disease could be treated; it is not a requirement that CNN-4 be mutated or abnormal. Rather, the overexpression of CNN-4 may actually overcome other defects within the cell. Thus, it is contemplated that a wide variety of hypeφroliferative diseases may be treated using CNN-4 therapy, including cancers of the brain, lung, liver, spleen, kidney, lymph node, pancreas, small intestine, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow, blood or other tissue.
In many contexts, it is not necessary that the diseased cell be killed or induced to undergo normal cell death or "apoptosis." Rather, to accomplish a meaningful treatment, all that is required is that the growth be slowed to some degree. It may be that the growth is completely blocked, however, or that some killing of cells within a hypeφroliferative tissue is achieved. Clinical terminology such as "remission" and
"reduction of tumor burden" also are contemplated given their normal usage. A. Genetic Based Therapies
One of the therapeutic embodiments contemplated by the present inventors is the intervention, at the molecular level, in the events involved in the tumorigenesis of some cancers. Specifically, the present inventors intend to provide, to a cancer cell, an expression construct capable of providing CNN-4 to that cell. The lengthy discussion of expression vectors, and the genetic elements employed therein, is incoφorated into this section by reference. Particularly preferred expression vectors are viral vectors such as adenovirus, adeno-associated virus, heφesvirus, vaccinia virus and retrovirus. Also preferred is liposomally-encapsulated expression vector.
Those of skill in the art are well aware of how to apply gene delivery to in vivo and ex vivo situations. For viral vectors, one generally will prepare a viral vector stock. Depending on the kind of virus and the titer attainable, one will deliver 1 X 104, 1 X 105, 1 X 106, 1 X 107, 1 X 108, 1 X 109, 1 X 1010, 1 X 10n or 1 X 1012 infectious particles to the patient. Similar figures may be extrapolated for liposomal or other non-viral formulations by comparing relative uptake efficiencies. Formulation as a pharmaceutically acceptable composition is discussed below.
Various routes are contemplated for various diseases. The section below on routes contains an extensive list of possible routes. For practically any disease, systemic delivery is contemplated. This will prove especially important for attacking microscopic or metastatic cancer. Where discrete tissue mass may be identified, a variety of direct, local and regional approaches may be taken. For example, the tumor may be directly injected with the expression vector. A tumor bed may be treated prior to, during or after resection. Following resection, one generally will deliver the vector by a catheter left in place following surgery. One may utilize the tumor vasculature to introduce the vector into the tumor by injecting a supporting vein or artery. A more distal blood supply route also may be utilized.
In a different embodiment, ex vivo gene therapy is contemplated. This approach is particularly suited, although not limited, to treatment of bone marrow associated diseases. In an e vivo embodiment, cells from the patient are removed and maintained outside the body for at least some period of time. During this period, a therapy is delivered, after which the cells are reintroduced into the patient; hopefully, any hypeφroliferative cells in the sample have been killed.
Autologous bone marrow transplant (ABMT) is an example of ex vivo gene therapy. Basically, the notion behind ABMT is that the patient will serve as his or her own bone marrow donor. Thus, a normally lethal dose of irradiation or chemotherapeutic may be delivered to the patient to kill tumor cells, and the bone marrow repopulated with the patients own cells that have been maintained (and perhaps expanded) ex vivo. Because, bone marrow often is contaminated with tumor cells, it is desirable to purge the bone marrow of these cells. Use of gene therapy to accomplish this goal is yet another way CNN-4 may be utilized according to the present invention.
B. Immunotherapies
Immunotherapeutics, generally, rely on the use of immune effector cells and molecules to target and destroy cancer cells. The immune effector may be, for example, an antibody specific for some marker on the surface of a tumor cell. The antibody alone may serve as an effector of therapy or it may recruit other cells to actually effect cell killing. The antibody also may be conjugated to a drug or toxin
(chemotherapeutic, radionuclide, ricin A chain, cholera toxin, pertussis toxin, etc.) and serve merely as a targeting agent. Alternatively, the effector may be a lymphocyte carrying a surface molecule that interacts, either directly or indirectly, with a tumor cell target. Various effector cells include cytotoxic T cells and NK cells.
C. Protein Therapy
Another therapy approach is the provision, to a subject, of CNN-4 polypeptide, active fragments, synthetic peptides, mimetics or other analogs thereof. The protein may be produced by recombinant expression means or, if small enough, generated by an automated peptide synthesizer. Formulations would be selected based on the route of administration and puφose including, but not limited to, liposomal formulations and classic pharmaceutical preparations. D. Combined Therapy with Immunotherapy, Traditional Chemo- or Radiotherapy
To kill cells, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the hypeφroliferative phenotype of cells, using the methods and compositions of the present invention, one may contact a "target" cell with a CNN-4 expression construct and at least one other agent. These compositions would be provided in a combined amount effective to kill or inhibit proliferation of the cell. This process may involve contacting the cells with the expression construct and the agent(s) or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the expression construct and the other includes the agent.
Alternatively, the gene therapy treatment may precede or follow the other agent treatment by intervals ranging from minutes to weeks to months. In embodiments where the other agent and expression construct are applied separately to the cell, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent and expression construct would still be able to exert an advantageously combined effect on the cell. In such instances, it is contemplated that one would contact the cell with both modalities within about 12-24 hours of each other and, more preferably, within about 6-12 hours of each other, with a delay time of only about 12 hours being most preferred. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (2, 3, 4, 5, 6 or
7) to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
It also is conceivable that more than one administration of either CNN-4 or the other agent will be desired. Various combinations may be employed, where CNN-4 is "A" and the other agent is "B", as exemplified below: A/B/A B/A B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
A/A/B/B A B/A/B A/B/B/A B/B/A/A B/A B/A B/A/A B B/B/B/A
A/A/A/B B/A/A/A A/B/A A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
Other combinations are contemplated. Again, to achieve cell killing, both agents are delivered to a cell in a combined amount effective to kill the cell.
Agents or factors suitable for use in a combined therapy are any chemical compound or treatment method that induces DNA damage when applied to a cell. Such agents and factors include radiation and waves that induce DNA damage such as, γ-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions, and the like. A variety of chemical compounds, also described as "chemotherapeutic agents," function to induce DNA damage, all of which are intended to be of use in the combined treatment methods disclosed herein. Chemotherapeutic agents contemplated to be of use, include, e.g., adriamycin, 5-fluorouracil (5FU), etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, and cisplatin (CDDP). The invention also encompasses the use of a combination of one or more DNA damaging agents, whether radiation-based or actual compounds, such as the use of X-rays with cisplatin or the use of cisplatin with etoposide. In certain embodiments, the use of cisplatin in combination with a CNN-4 expression construct is particularly preferred as this compound.
In treating cancer according to the invention, one would contact the tumor cells with an agent in addition to the expression construct. This may be achieved by irradiating the localized tumor site with radiation such as X-rays, UV-light, γ-rays or even microwaves. Alternatively, the tumor cells may be contacted with the agent by administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a compound such as, adriamycin, 5-fluorouracil, etoposide, camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin. The agent may be prepared and used as a combined therapeutic composition, or kit, by combining it with a CNN-4 expression construct, as described above. In addition to combining CNN-4-targeted therapies with chemo- and radiotherapies, it also is contemplated that combination with other gene therapies will be advantageous. For example, targeting of CNN-4 and p53, Rb, p21 or plό mutations at the same time may produce an improved anti-cancer treatment. Any other tumor-related gene conceivably can be targeted in this manner, for example, p21, p53, Rb, APC, DCC, NF-1, NF-2, BCRA2, pi 6, FHIT, WT-1, MEN-I, MEN-LI, BRCA1, VHL, FCC, MCC, ras, myc, neu, raf, erb, src, fins, jun, trk, ret, gsp, hst, bcl and abl.
IX. Method of Stimulating Cell Growth
In still another embodiment, the present invention presents the opportunity to intervene therapeutically in clinical states where a lack of cellular growth either is at the cause, or prevents recover from, the state. Examples of such states include bone wasting disease or damage, nervous tissue degenerative disease or damage, hematopoietic cell disease, and connective tissue healing. By blocking CNN-4 function, and thereby promoting cell division, one may stimulate non- or poorly proliferative cells to divide.
The general techniques by which these methods can be applied are described elsewhere. For example, the general approaches by which CNN-4 function may be blocked are described in the section addressing the creating of cell lines. Transfer of proteins and genetic material also are described. Routes of administration and pharmaceutical formulations are described in the preceding section.
X. Methods of Selection
Another embodiment of the present invention involves use of CNN-4 as a selection marker for gene transfer studies. In this application, CNN-4 gene is inserted into an expression cassette. In preferred embodiments, the CNN-4 express cassette is covalently linked to a gene that the investigator wishes to transfer into a particular cell, which is either under control of the same or a different promoter, as compared to
CNN-4. A. Homologous Recombination
For homologous recombination studies, it is envisioned that the CNN-4 gene can be used as follows. The CNN-4 gene is placed at one end of a DNA construct that comprises a selected gene to be transferred, flanked by target gene sequences. The CNN-4 gene is under the control of a promoter that will express CNN-4 in the host cell, under suitable conditions, at a level that will kill the cell.
A target gene within a host cell is selected as the location into which a selected gene is to be transferred. Sequences homologous to the target gene are included in the expression construct, and the selected gene is inserted such that target gene homologous sequences are interrupted by the selected gene or, put another way, such the target gene homologous sequences "flank" the selected gene. In preferred embodiments, a drug selectable marker gene also is inserted into the target gene homologous sequences. Given this possibility, it should be apparent that the term "flank" is used broadly herein, namely, as describing target homologous sequences that are both upstream (5') and downstream (3') of the selected gene and/or the drug selectable marker gene. In effect, the flanking sequences need not directly abut the genes they "flank."
The construct for use in this embodiment is further characterized as having a functional CNN-4 gene attached thereto. Thus, one possible arrangement of sequences would be:
5'-CNN-4,flanking target sequences»selected gene'drug-selectable marker gene»flanking target sequences-3'
Of course, the CNN-4 could come at the 3'-end of the construct and the selected gene and drug-selectable marker genes could exchange positions.
The principal behind the use of such a vector is as follows. Many recombination events, following introduction of an expression vector into a host cell, are non-homologous. In certain applications, these non-homologous recombination events are undesirable and, thus, it is beneficial to select against these events. When using a vector as described above, a non-homologous recombination event likely will result in incoφoration of all the sequences, including the CNN-4 gene. Expression of CNN-4 will result in killing of non-homologous recombinants.
On the other hand, site-specific recombination, relying on the homology between the vector and the target gene, will result in incoφoration of the selected gene and the drug selectable marker gene only; CNN-4 sequences will not be introduced in the homologous recombination event because they lie outside the flanking sequences. This double-selection procedure (drugres/CNN-4") should yield recombinants that lack the target gene and express the selected gene. Further screens for these phenotypes, either functional or immunologic, may be applied.
A modification of this procedure is one where no selected gene is included, i.e., only the selectable marker is inserted into the target gene homologous sequences.
Use of this kind of construct will result in the "knock-out" of the target gene only. Again, proper recombinants are screened by drug resistance and CNN-4" phenotype.
Examples of processes that use negative selection to enrich for homologous recombinants include the disruption of targeted genes in embryonic stem cells or transformed cell lines (Mortensen, 1993, Willnow & Herz, 1994) and the production of recombinant virus such as adenovirus (Imler et al, 1995).
B. Non-Homologous Recombination In other embodiments, the CNN-4 gene can be used as a tool for screening for transfer of expression vectors in a non-homologous fashion. In this case, the expression construct contains the selected gene that is to be delivered, a drug selectable marker gene and a CNN-4 cDNA or genomic clone. Preferably, the selected gene is inteφosed between the CNN-4 transporter gene and the marker gene.
The principle behind the selection process is as follows. While some recombinants may only achieve partial integration of the construct, i.e., the drug selectable marker or the CNN-4 sequences, a cell expressing products of both these vector genes also likely contains the intervening sequences, namely, the selected gene. Thus, following recombination, the cells are screened for drug resistance. Of the remaining cells, these are screened for CNN-4 expression, i.e., death under suitable conditions. If the cell satisfies these double-selection criteria, they should also expression the selected gene.
The drug screen is a conventional positive selection protocol where the surviving cells are those desired. The CNN-4 screen may be accomplished by utilizing an inducible promoter to control the expression of CNN-4. In an uninduced state, no CNN-4 is produced. However, using the proper signal, CNN-4 synthesis may be turned no and, hence, cells that contain this gene can be identified. Being a negative selection process, this approach generally involves preparing replica or duplicate cultures, where a first and a second cell culture of the drug-resistant cells are prepared. One cell culture is contacted with the inducer, and cells are identified that are CNN-4+, one identifies replicas that died under induction. Further characterization of these doubly-selected clones should confirm expression of the selected gene.
Examples of conventional antibiotic resistance markers are those conferring resistance to neomycin, hygromycin, puromycin or zeocin; and xanthine-guanine phosphoribosyl transferase, HisD and dihydrofolate reductase genes. The operation of drug selection or antibiotic resistance marker genes is well known to those of skill in the art.
XI. Methods of Identifying CNN-4 Homologues
While homologues of CNN-4 from other species of Drosophila have readily been found, attempts to identify homologues of CNN-4 in other organisms using direct nucleic acid hybridization on whole genome Southern blots using the CNN-4 cDNA as a probe has not been unsuccessful. It is likely that non-specificity of the probe is preventing identification. This can be circumvented as follows. The CNN-4 locus will be cloned from the species of Drosophila that we have identified as having a recognizable copy of the gene. These include D. simulans, D. saltans, D. willistoni, D. funebris, D. virilis and D. hydei. The cloned fragments from these species will be sequenced and their coding potential determined. Sequence comparisons among the various species will reveal those portion of the CNN-4 open reading frame that are the most highly conserved. From these sequences, probes or PCR primers will be designed that will hybridize specifically with the CNN-4 homologues in more distantly related species. This process can be repeated with the CNN-4 from each new organism to refine the conserved nature of the probes and primers. Just such a protocol has worked for the recovery of homologous mammalian genes that were originally found in model systems such as Drosophila. Goodrich et al (1996).
XII. Transgenic Organisms In one embodiment of the invention, transgenic animals are produced which contain a transgene encoding a functional CNN-4 polypeptide or variants thereof. Transgenic animals expressing CNN-4 transgenes, recombinant cell lines derived from such animals and transgenic embryos may be useful in methods for screening for and identifying agents that induce or repress function of CNN-4. Transgenic animals of the present invention also can be used as models for studying indications such as cancers. In cells where CNN-4 is lethal, it will be desirable to place the CNN-4 gene under the control of an inducible promoter. Selected animals include fruit flies, mice, rats, sheep, guinea pigs, cows or other suitable organism.
In one embodiment of the invention, a CNN-4 transgene is introduced into a non-human host to produce a transgenic animal expressing a CNN-4 product. The transgenic animal is produced by the integration of the transgene into the genome in a manner that permits the expression of the transgene. Methods for producing transgenic animals are generally described by Wagner and Hoppe (U.S. Patent 4,873,191; which is incoφorated herein by reference), Brinster et al. 1985; which is incoφorated herein by reference in its entirety) and in "Manipulating the Mouse Embryo; A Laboratory Manual" 2nd edition (eds., Hogan, Beddington, Costantimi and Long, Cold Spring Harbor Laboratory Press, 1994; which is incoφorated herein by reference in its entirety).
It may be desirable to replace the endogenous CNN-4 gene by homologous recombination between the transgene and the endogenous gene; or the endogenous gene may be eliminated by deletion as in the preparation of "knock-out" animals. Typically, a CNN-4 gene flanked by genomic sequences is transferred by microinjection into a fertilized egg. The microinjected eggs are implanted into a host female, and the progeny are screened for the expression of the transgene. Transgenic animals may be produced from the fertilized eggs from a number of animals including, but not limited to reptiles, amphibians, birds, mammals, and fish. Within a particularly preferred embodiment, transgenic mice are generated which overexpress CNN-4 (e.g., inducibly) or express a mutant form of the CNN-4 polypeptide. Alternatively, the absence of a CNN-4 in "knock-out" mice permits the study of the effects that loss of CNN-4 protein has on a cell in vivo. Knock-out mice also provide a model for the development of CNN-4-related hypeφroliferative diseases.
As noted above, transgenic animals and cell lines derived from such animals may find use in certain testing experiments. In this regard, transgenic animals and cell lines capable of expressing wild-type or mutant CNN-4 may be exposed to test substances. These test substances can be screened for the ability to enhance wild-type CNN-4 expression and or function or impair the expression or function of mutant CNN-4.
XIII. Pharmaceutical Preparations and Routes of Administration
Where clinical applications are contemplated, it will be necessary to prepare pharmaceutical compositions - expression vectors, 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 absoφtion of the 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 absoφtion 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 incoφorated 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 incoφorating 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. 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.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While some of the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein 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 of the invention as defined by the appended claims. IX. REFERENCES
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Claims

1. An isolated CNN-4 polypeptide.
2. The isolated polypeptide of claim 1, wherein said CNN-4 is vertebrate CNN-4.
3. The isolated polypeptide of claim 1, wherein said CNN-4 is mammalian CNN-
4.
4. The isolated polypeptide of claim 1, wherein said CNN-4 is Drosophila melanogaster CNN-4.
5. The isolated polypeptide of claim 4, wherein said CNN-4 has the sequence of SEQ LD NO:2.
6. The isolated polypeptide of claim 1, wherein said CNN-4 is human CNN-4.
7. An isolated peptide having between about 10 and about 50 consecutive residues of CNN-4.
8. The isolated peptide of claim 7, wherein said CNN-4 has the sequence of SEQ
LD NO:2.
9. The isolated peptide of claim 7, wherein said peptide has about 15 consecutive
residues of CNN-4.
10. The isolated peptide of claim 7, wherein said peptide has about 20 consecutive
residues of CNN-4.
11. The isolated peptide of claim 7, wherein said peptide has about 25 consecutive
residues of CNN-4.
12. The isolated peptide of claim 7, wherein said peptide has about 30 consecutive
residues of CNN-4.
13. The isolated peptide of claim 7, wherein said peptide has about 35 consecutive
residues of CNN-4.
14. The isolated peptide of claim 7, wherein said peptide has about 40 consecutive
residues of CNN-4.
15. The isolated peptide of claim 7, wherein said peptide has about 50 consecutive
residues of CNN-4.
16. The isolated peptide of claim 7, wherein said peptide is about 10 to about 100
residues in length.
17. The isolated peptide of claim 7, wherein said peptide is about 10 residues in length.
18. The isolated peptide of claim 7, wherein said peptide is about 15 residues in
length.
19. The isolated peptide of claim 7, wherein said peptide is about 20 residues in
length.
20. The isolated peptide of claim 7, wherein said peptide is about 25 residues in
length.
21. The isolated peptide of claim 7, wherein said peptide is about 30 residues in
length.
22. The isolated peptide of claim 7, wherein said peptide is about 40 residues in
length.
23. The isolated peptide of claim 7, wherein said peptide is about 50 residues in
length.
24. The isolated peptide of claim 7, wherein said peptide is conjugated to a carrier
molecule.
25. The isolated peptide of claim 24, wherein the carrier molecule is selected from
the group consisting of KLH and BSA.
26. A monoclonal antibody that binds immunologically to CNN-4.
27. The monoclonal antibody of claim 26, wherein said antibody is species
specific.
28. The monoclonal antibody of claim 26, wherein said antibody further comprises
a detectable label.
29. The monoclonal antibody of claim 28, wherein the label is selected from the group consisting of a fluorescent label, a chemilluminescent label, a radiolabel
and an enzyme.
30. A hybridoma cell that produces a monoclonal antibody that binds
immunologically to CNN-4.
31. A polyclonal antisera, antibodies of which bind immunologically to CNN-4.
32. An isolated polynucleotide encoding CNN-4, or the complement thereof.
33. The isolated polynucleotide of claim 32, wherein said CNN-4 is vertebrate
CNN-4.
34. The isolated polynucleotide of claim 32, wherein said CNN-4 is mammalian
CNN-4.
35. The isolated polynucleotide of claim 32, wherein said CNN-4 is Drosophila
melanogaster CNN-4.
36. The isolated polynucleotide of claim 35, wherein said CNN-4 has the amino
acid sequence of SEQ ID NO:2.
37. The isolated polynucleotide of claim 35, wherein said CNN-4 has the
polynucleotide sequence of SEQ LD NO: 1, or the complement thereof.
38. The isolated polynucleotide of claim 32, wherein said CNN-4 is human CNN-
4.
39. The isolated polynucleotide of claim 32, wherein said polynucleotide is
selected from the group consisting of genomic DNA, complementary DNA
and RNA.
40. An expression cassette comprising a DNA segment encoding CNN-4 and a
promoter operably linked to said segment.
41. The expression cassette of claim 40, further comprising a polyadenylation signal operably linked to said segment.
42. The expression cassette of claim 40, wherein said promoter is a tissue specific promoter.
43. The expression cassette of claim 40, wherein said promoter is an inducible promoter.
44. The expression cassette of claim 40, wherein said promoter is a constitutive promoter.
45. A replicable vector comprising an expression cassette comprising (i) a DNA
segment encoding CNN-4 and a promoter operably linked to said segment and (ii) an origin of replication.
46. The replicable vector of claim 45, wherein said vector is a viral vector selected
from the group consisting of retrovirus, adenovirus, heφesvirus, vaccinia
virus, polyoma virus and adeno-associated virus.
47. The replicable vector of claim 46, wherein said viral vector is packaged in a
virus particle.
48. The replicable vector of claim 45, wherein said vector is packaged in a
liposome.
49. The replicable vector of claim 45, wherein the vector is of a size selected from
the group consisting of about 5000 bases, about 10,000 bases, about 15,000
bases, about 20,000 bases, about 25,000 bases, about 30,000 bases, about
35,000 bases, about 40,000 bases, about 45,000 bases, about 50,000 bases,
about 75,000 bases and about 100,000 bases.
50. An isolated oligonucleotide of between about 10 and about 100 bases, the sequence of which comprises between about 10 and 100 consecutive bases of
SEQ ID NO: 1.
51. The oligonucleotide of claim 50, wherein the oligonucleotide is 10 bases in
length.
52. The oligonucleotide of claim 50, wherein the oligonucleotide is about 15 bases
in length.
53. The oligonucleotide of claim 50, wherein the oligonucleotide is about 20 bases
in length.
54. The oligonucleotide of claim 50, wherein the oligonucleotide is about 30 bases
in length.
55. The oligonucleotide of claim 50, wherein the oligonucleotide is about 40 bases
in length.
56. The oligonucleotide of claim 50, wherein the oligonucleotide is about 50 bases
in length.
57. The oligonucleotide of claim 50, wherein the oligonucleotide is about 75 bases
in length.
58. The oligonucleotide of claim 50, wherein the oligonucleotide is about 100
bases in length.
59. A method for expressing a CNN-4 polypeptide comprising the steps of:
(i) providing a cell;
(ii) introducing into said cell an expression cassette comprising a DNA
segment encoding a CNN-4 polypeptide and a promoter
operably linked to said segment; and
(iii) culturing said cell under conditions permitting expression of CNN-4.
60. A method for identifying a CNN-4-binding protein comprising the steps of: (i) providing a CNN-4 polypeptide;
(ii) contacting said CNN-4 polypeptide with a sample; and
(iii) identifying a polypeptide bound to said CNN-4 polypeptide.
61. The method of claim 60, wherein said method comprises a two-hybrid
selection system.
62. A method of isolating a CNN-4 related gene comprising the steps of:
(i) selecting a probe consisting essentially of conserved regions from
CNN-4;
(ii) hybridizing said probe to RNA or DNA from a candidate cell; and
(iii) isolating the hybridizing RNA or DNA.
63. The method of claim 62, further comprising cloning said hybridizing DNA.
64. A method of selecting a cell comprising the steps of:
(i) transforming a population of cells with an expression cassette
comprising a DNA segment encoding a CNN-4 and a promoter operably linked to said segment; (ii) culturing cells under conditions suitable for the expression of CNN-4;
and
(iii) selecting a cell that survives step (ii).
65. The method of claim 64, wherein said expression cassette is part of a larger
polynucleotide further comprising a second gene.
66 The method of claim 65, wherein said second gene is flanked by genomic
sequences of a target site in said cell, and said CNN-4 gene is not flanked by said genomic sequences.
67. A method for selecting a cell comprising: (i) transforming a population of cells with an expression cassette
comprising a DNA segment encoding CNN-4 and a promoter
operably linked to said segment;
(ii) culturing cells under conditions suitable for the expression of CNN-4;
and
(iii) selecting a cell is killed in step (ii).
68. The method of claim 67, wherein said promoter is inducible and said
conditions result in the induction of said promoter.
69. The method of claim 68, further comprising a step, between steps (i) and (ii),
of making a replicate culture of transformed cells.
70. The method of claim 67, wherein said expression cassette is part of a larger
polynucleotide further comprising a second gene.
71. The method of claim 66, wherein said second gene is a selectable marker.
72. The method of claim 67, wherein said selectable marker confers resistance to neomycin, hygromycin, puromycin, zeocin, mycophenolic acid, histidinol or
methotrexate.
73. A method of diagnosing a hypeφroliferative disease comprising the steps of:
(i) obtaining a sample from a subject; and
(ii) assaying CNN-4 in cells of said sample.
74. The method of claim 73, wherein said hypeφroliferative disease is cancer.
75. The method of claim 74, wherein said cancer is selected from the group
consisting of brain, lung, liver, spleen, kidney, lymph node, small intestine,
pancreas, blood cells, colon, stomach, breast, endometrium, prostate, testicle, ovary, skin, head and neck, esophagus, bone marrow and blood cancer.
76. The method of claim 73, wherein said assaying comprises measuring CNN-4
mRNA levels in said sample.
77. The method of claim 83, wherein said assaying comprises measuring CNN-4
protein levels in said sample.
78. The method of claim 77, wherein said measuring comprises an immunoassay.
79. The method of claim 73, wherein said assaying comprises characterizing the
structure of a CNN-4 polynucleotide in said sample.
80. The method of claim 79, wherein said characterizing comprises sequencing.
81. The method of claim 73, further comprising the step of comparing the CNN-4
of said sample with that of a non-diseased sample.
82. A method for altering the hypeφroliferative phenotype of a cell comprising the
step of contacting the cell with CNN-4 under conditions permitting the uptake of CNN-4 by said cell.
83. The method of claim 82, wherein the a phenotype is selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth,
metastasis and blocking mitosis.
84. The method of claim 93, wherein said CNN-4 is encapsulated in a liposome.
85. A method for altering the phenotype of a hypeφroliferative cell comprising the
step of introducing into said cell an expression cassette comprising (i) a DNA
segment encoding CNN-4 and (ii) a promoter active in cell, wherein said
promoter is operably linked to said segment, under conditions permitting the
uptake of said expression cassette by said hypeφroliferative cell.
86. The method of claim 85, wherein the a phenotype is selected from the group consisting of proliferation, migration, contact inhibition, soft agar growth
metastasis and blocking mitosis.
87. The method of claim 85, wherein said expression cassette is encapsulated in a
liposome.
88. The method of claim 85, wherein said expression cassette is contained in a
viral vector selected from the group consisting of retrovirus, adenovirus,
adeno-associated virus, vaccinia virus and heφesvirus.
89. The method of claim 88, wherein said viral vector is packaged in a viral
particle.
90. A method for treating a hypeφroliferative disease in a subject comprising the
step of introducing into a hypeφroliferative cell in said subject a CNN-4
polypeptide.
91. A method for treating a hypeφroliferative disease in a subject comprising the
step of introducing into a hypeφroliferative cell in said subject an expression cassette comprising (i) a DNA segment encoding CNN-4 and (ii) a promoter
active in cell, wherein said promoter is operably linked to said segment, under
conditions permitting the uptake of said nucleic acid by said tumor cell.
92. A non-human transgenic organism, the cells of which comprise a transgenic
CNN-4 gene.
93. The organism of claim 92, wherein the transgenic CNN-4 gene is heterologous
to said organism.
94. The organism of claim 92, wherein the transgenic CNN-4 gene is homologous
to said organism.
95. The organism of claim 92, wherein the transgenic CNN-4 gene is under the
control of a promoter.
96. The organism of claim 95, wherein the promoter is a tissue specific promoter.
97. The organism of claim 95, wherein the promoter is an inducible promoter.
98. The organism of claim 92, wherein said CNN-4 is a mutant CNN-4 gene.
99. The organism of claim 92, wherein both endogenous copies of CNN-4 are
dysfunctional.
100. The organism of claim 92, wherein the organism is a vertebrate.
101. The organism of claim 92, wherein the organism is a mammal.
102. The organism of claim 92, wherein the organism is from the species
Drosophila.
103. The organism of claim 92, wherein the organism is a mouse.
104. A method of screening a candidate substance for proliferative activity
comprising the steps of:
(i) providing a cell lacking functional CNN-4; (ii) contacting said cell with said candidate substance; and
(iii) determining the effect of said candidate substance on said cell.
105. The method of claim 104, wherein said determining comprises comparing one
or more characteristics of the cell in the presence of said candidate substance
with characteristics of a cell in the absence of said candidate substance.
106. The method of claim 105, wherein said characteristic is selected from the
group consisting of CNN-4 expression, proliferation, metastasis, contact
inhibition, soft agar growth, blocking mitosis, tumor formation, tumor
progression and tissue invasion.
107. The method of claim 104, wherein said cell is contacted in vitro.
108. The method of claim 104, wherein said cell in contacted in vivo.
109. A method for increasing proliferation of cellular population comprising the
step of inhibiting CNN-4 function in at least one cell of said population.
110. The method of claim 109, wherein inhibition is achieved by introducing into a
cell of said population an expression cassette comprising (i) a DNA segment
encoding at least a portion of a CNN-4 complementary sequence and (ii) a
promoter active in cell, wherein said promoter is operably linked to said DNA
segment, under conditions permitting the uptake of said nucleic acid by said
tumor cell.
111. The method of claim 110, wherein said DNA segment is complementary to an
exon of CNN-4.
112. The method of claim 110, wherein said DNA segment is complementary to an
intron of CNN-4.
113. The method of claim 110, wherein said DNA segment is complementary to adjacent intronic and exonic regions of CNN-4.
114. The method of claim 110, wherein said DNA segment is complementary to a
splice junction of CNN-4.
115. The method of claim 110, wherein said DNA segment is complementary to a
translation initiation site of CNN-4.
116. The method of claim 109, wherein inhibition is achieved by introducing into a
cell of said population an expression cassette comprising (i) a DNA segment
encoding a single-chain antibody that binds immunologically to CNN-4 and
(ii) a promoter active in cell, wherein said promoter is operably linked to said
DNA segment, under conditions permitting the uptake of said nucleic acid by said tumor cell.
PCT/US1999/029251 1998-12-11 1999-12-08 Compositions and methods relating to a new cell cycle regulating gene WO2000034524A1 (en)

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WO (1) WO2000034524A1 (en)

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LI ET AL: "The homeotic target gene centrosomin encodes an essential centrosomal component", CELL, vol. 85, 17 May 1996 (1996-05-17), pages 585 - 596, XP002927582 *

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