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WO2000020579A1 - Forme epissee de l'oncogene erbb-2/neu - Google Patents

Forme epissee de l'oncogene erbb-2/neu Download PDF

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Publication number
WO2000020579A1
WO2000020579A1 PCT/CA1999/000912 CA9900912W WO0020579A1 WO 2000020579 A1 WO2000020579 A1 WO 2000020579A1 CA 9900912 W CA9900912 W CA 9900912W WO 0020579 A1 WO0020579 A1 WO 0020579A1
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Prior art keywords
protein
erbb
splice
nucleic acid
substance
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PCT/CA1999/000912
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English (en)
Inventor
William J. Muller
Peter M. Siegel
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Mcmaster University
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Application filed by Mcmaster University filed Critical Mcmaster University
Priority to AU59636/99A priority Critical patent/AU5963699A/en
Publication of WO2000020579A1 publication Critical patent/WO2000020579A1/fr

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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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the invention relates to a novel spliced form of gene that encodes a constitutively tyrosine phosphorylated erbB-2 receptor.
  • the invention includes nucleic acid molecules encoding the protein and truncations, analogs, and homologs of the protein; and uses of the protein and nucleic acid molecules in controlling cell transformation and cancer.
  • Activation of growth factor receptor tyrosine kinases has been implicated in the malignant progression of a number of different tumor types.
  • elevated expression of the erbB-2 oncogene has been observed in a significant proportion of human breast and ovarian cancers (Slamon et at, 1987; 1989).
  • the extent of overexpression of erbB-2 in lymph node positive breast cancer patients has been inversely correlated with patient survival (reviewed in Hynes and Stern, 1994).
  • Direct evidence for the importance of this gene in the induction of mammary tumors has been obtained from studies of transgenic mice that express either an activated or wild-type neu cDNA in the mammary epithelium.
  • Mammary epithelial expression of the activated version of neu results in the rapid induction of multifocal mammary tumors in every female transgenic animal (Muller et al, 1988; Guy e t al., 1996).
  • mammary epithelial expression of the neu proto-oncogene leads to development of focal mammary tumors which develop after a longer latency period (Guy e t al., 1992).
  • these wild-type neu transgenic strains frequently developed secondary metastatic lesions in the lung (Guy et al, 1992).
  • the present inventors have identified a spliced form of mRNA which translates into an erbB-2 receptor which has increased catalytic activity capable of transforming Rat-1 fibroblasts.
  • the increased catalytic activity is reflected by increased tyrosine phosphorylation of the receptor.
  • the increase in catalytic activity of this receptor may constitute a significant causative factor in the genesis of human breast tumors.
  • the present invention therefore provides a purified and isolated nucleic acid molecule (SPLICE) comprising a sequence encoding a constitutively tyrosine phosphorylated erbB-2 receptor, which is the protein of the invention, having increased catalytic activity and capable of transforming Rat-1 fibroblasts and which may generally be referred to as SPLICE erbB-2 protein.
  • the purified and isolated nucleic acid molecule comprises: (i) a nucleic acid sequence encoding a SPLICE erbB-2 protein having the amino acid sequence as shown in Figure 2 and SEQ ID NO:2; and, (ii) nucleic acid sequences complementary to (i).
  • the purified and isolated nucleic acid molecule comprises:
  • the invention also contemplates an isolated and purified (a) nucleic acid molecule comprising a sequence encoding a truncation of SPLICE erbB-2, an analog, homolog or truncation thereof; (b) nucleic acid molecule comprising a sequence which hybridizes under high stringency conditions to the nucleic acid encoded by a SPLICE erbB-2 protein having the amino acid sequence as shown in Figure 2 or SEQ ID NO:2, wherein T can also be U, or complementary sequences thereto, or by a related protein; and (c) nucleic acid molecule comprising a sequence which hybridizes under high stringency conditions to the nucleic acid sequence as shown in Figure 1, wherein T can also be U, or complementary sequences thereto.
  • the invention further contemplates a purified and isolated double stranded nucleic acid molecule containing a nucleic acid molecule of the invention, hydrogen bonded to a complementary nucleic acid base sequence.
  • the nucleic acid molecules of the invention may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted coding sequence.
  • recombinant expression vectors adapted for transformation of a host cell preferably mammary cells, may be constructed which comprise a nucleic acid molecule of the invention and one or more transcription and translation elements operatively linked to the nucleic acid molecule.
  • the recombinant expression vector can be used to prepare transformed host cells expressing a SPLICE erbB-2 protein of the invention or a related protein. Therefore, the invention further provides host cells containing a recombinant molecule of the invention.
  • the invention also contemplates transgenic non-human mammals whose germ cells and somatic cells contain a recombinant molecule comprising a nucleic acid molecule of the invention which encodes a SPLICE erbB-2 protein or an analog thereof, i.e., the protein with an insertion, substitution or deletion mutation.
  • the invention further provides a method for preparing a SPLICE protein, or a related protein utilizing the purified and isolated nucleic acid molecules of the invention.
  • a method for preparing a SPLICE erbB-2 protein comprising (a) transferring a recombinant expression vector of the invention into a host cell; (b) selecting transformed host cells from untransformed host cells; (c) culturing a selected transformed host cell under conditions which allow expression of the SPLICE erbB-2 protein; and (d) isolating the SPLICE erbB-2 protein.
  • the invention further broadly contemplates a purified and isolated SPLICE erbB-2 protein which is capable of transforming Rat-1 fibroblasts.
  • the SPLICE erbB-2 protein has an in frame deletion of sixteen (16) amino acids, two of which are conserved cysteine residues and an approximate molecular weight of 50KD.
  • a purified SPLICE erbB-2 protein is provided which has the amino acid sequence as shown in Figure 2 or SEQ ID NO:2.
  • the invention also includes truncations of the protein and analogs, homologs, and isoforms of the protein and truncations thereof.
  • the SPLICE erbB-2 proteins of the invention may be conjugated with other molecules, such as proteins to prepare fusion proteins. This may be accomplished, for example, by the synthesis of N-terminal or C-terminal fusion proteins.
  • the invention further contemplates antibodies having specificity against an epitope of a SPLICE erbB-2 protein of the invention.
  • Antibodies may be labelled with a detectable substance and they may be used to detect the SPLICE erbB-2 protein of the invention in tissues and cells.
  • the invention also permits the construction of nucleotide probes which are unique to the nucleic acid molecules of the invention and accordingly to a SPLICE erbB-2 protein of the invention.
  • the invention also relates to a probe comprising a sequence encoding a SPLICE erbB-2 protein or fragment thereof.
  • the probe may be labelled, for example, with a detectable substance and it may be used to select from a mixture of nucleotide sequences a nucleotide sequence coding for a protein which displays one or more of the properties of the protein of the invention.
  • the probes may also be used to detect a nucleic acid encoding a SPLICE erbB-2 protein of the invention in tissues and cells. In particular, the probes may be used as a diagnostic tool on biopsied tissues to assess the transformed state of the cell.
  • the SPLICE erbB-2 protein is likely involved in the induction of human breast tumors. Accordingly, in one embodiment the present invention provides a method of modulating SPLICE erbB-2 activity comprising administering an effective amount of inhibitor of SPLICE erbB-2 protein activity to a cell or animal in need thereof. In such an embodiment, it may be desirable to inhibit the expression of the SPLICE erbB-2 protein. Accordingly, the present invention provides a method of inhibiting or reducing cell transformation comprising administering an agent that inhibits the expression or activity of the SPLICE erbB-2 protein to an animal or cell in need thereof. Agents that may inhibit the SPLICE erbB-2 protein include antibodies to the protein or antisense oligonucleotides complimentary to a portion of a nucleic acid sequence encoding the protein.
  • the invention still further provides a method for identifying a substance which is capable of binding to a SPLICE erbB-2 protein or inactive form thereof, comprising reacting the SPLICE erbB-2 protein, or inactivated form, with at least one substance which potentially can bind with the SPLICE erbB-2 protein or an inactivated form thereof, under conditions which permit the formation of complexes between the substance and the protein or an inactivated form thereof, and assaying for complexes, for free substance, for non-complexed SPLICE erbB-2 pr inactivated form or for activation of SPLICE erbB-2
  • a yeast two hybrid assay system may be utilized as a method for identifying proteins which interact with the protein (Fields, S.
  • the invention provides a method for assaying a medium for the presence of an agonist or antagonist of the interaction of a SPLICE erbB-2 protein or an activated form thereof, and a substance which binds to the protein or an activated form thereof.
  • the method comprises providing a known concentration of a SPLICE erbB-2 protein, with a substance which is capable_of binding to the protein and a suspected agonist or antagonist substance under conditions which permit the formation of complexes between the substance and SPLICE erbB-2 protein, and assaying for complexes, free substance, non-complexed SPLICE erbB-2 protein, or for activation of the protein.
  • Substances which affect the SPLICE erbB-2 protein may also be identified using the methods of the invention by comparing the pattern and level of expression of the protein in tissues and cells in the presence, and in the absence of the substance.
  • Antibodies to SPLICE erbB-2 protein or antisense oligonucleotides complimentary to a nucleic acid encoding the SPLICE erbB-2 protein as well as substances identified using a method of the invention may be used in the treatment of conditions involving cell transformation such as cancer. Accordingly, the substances may be formulated into pharmaceutical compositions for adminstration to individuals suffering from cancer and preferably breast cancer. Accordingly, the substances may be formulated into pharmaceutical compositions for administration to individuals suffering from cancer.
  • Figure 1 shows the cDNA sequence of the SPLICE erbB-2 protein of the invention.
  • Figure 2 shows the amino acid sequence of SPLICE erbB-2 protein.
  • Figure 3a provides a schematic representation of a small region of the human erbB-2 genomic locus indicating the exon-intron boundaries.
  • Figure 3b shows an alignment of the wild-type and alternatively spliced erbB-2 messages indicating the sequences removed by the aberrant splicing event (grey box).
  • Figure 3c shows alternate mRNA splicing results in an erbB-2 transcript harboring an in-frame deletion.
  • Figure 4a shows partial amino acid sequence alignment of the wild-type and altered erbB-2 receptors tested in Rat-1 focus assays.
  • Figure 4b shows representative plates from focus assay #1 (see Table 1) illustrating the relative transforming abilities of the various forms of erbB-2 indicated in Figure 4a.
  • Figure 5a shows immunoblots of lysates from stable cell lines expressing wild-type ErbB-2 (WT) or the three altered receptors (VE, ECD. SPLICE).
  • Figure 5b shows immunoblots of lysates from stable cell lines expressing the various ErbB-2 receptors.
  • A Ala - alanine
  • C Cys - cysteine
  • D Asp- aspartic acid
  • E Glu - glutamic acid
  • F Phe - phenylalanine
  • G Gly - glycine
  • H His - histidine
  • I He - isoleucine
  • K Lys - lysine
  • L Leu - leucine
  • M Met - methionine
  • N Asn - asparagine
  • P Pro - proline
  • Q Gin - glutamine
  • R Arg - arginine
  • S Ser - serine
  • T Thr - threonine
  • V Val - valine
  • W Trp- tryptophan
  • Y Tyr - tyrosine
  • p.Y. P.Tyr - phosphotyrosine.
  • the invention provides an isolated and purified nucleic acid molecule having a sequence encoding a SPLICE erbB-2 protein.
  • isolated and purified refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
  • nucleic acid is intended to include DNA and RNA and can be either double stranded or single stranded.
  • the nucleic acid sequence of the cDNA encoding the SPLICE erbB-2 protein is shown in Figure 1 and SEQ.ID.NO.:l.
  • the invention includes nucleic acid molecules encoding truncations of SPLICE erbB-2 protein, and analogs and homologs of the protein and truncations thereof, as described herein. It will further be appreciated that variant forms of nucleic acid molecules of the invention which arise by alternative splicing of an mRNA corresponding to a cDNA of the invention are encompassed by the invention.
  • nucleic acid molecule which hybridizes under high stringency conditions to a nucleic acid molecule which comprises a sequence which encodes SPLICE erbB-2 protein having the amino acid sequence shown in Figure 2.
  • Appropriate stringency conditions which promote DNA hybridization are known to those skilled in the art, or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, 6.0 x sodium chloride/ sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C may be employed.
  • the stringency may be selected based on the conditions used in the wash step.
  • the salt concentration in the wash step can be selected from a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be at high stringency conditions, at about 65°C.
  • nucleic acid molecules encoding a protein having the activity of SPLICE erbB-2 protein as described herein, and having a sequence which differs from the nucleic acid sequence shown in Figure 1, due to degeneracy in the genetic code are also within the scope of the invention.
  • DNA sequence polymorphisms within the nucleotide sequence of SPLICE erbB-2 protein may result in "silent" mutations in the DNA which do not affect the amino acid encoded.
  • DNA sequence polymorphisms may lead to changes in the amino acid sequences of the protein within a population. It will be appreciated by one skilled in the art that these variations in one or more nucleotides of the nucleic acids encoding proteins having the activity of the SPLICE erbB-2 protein may exist among individuals within a population due to natural allelic variation. Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of the invention.
  • An isolated and purified nucleic acid molecule of the invention which comprises DNA can be isolated by preparing a labelled nucleic acid probe based on all or part of the nucleic acid sequence shown in Figure 1 and using this labelled nucleic acid probe to screen an appropriate DNA library (e.g. a cDNA or genomic DNA library).
  • a cDNA library made from lymphocytes such as B cells can be used to isolate a cDNA encoding a protein having SPLICE erbB-2 activity by screening the library with the labelled probe using standard techniques.
  • a genomic DNA library can be similarly screened to isolate a genomic clone encompassing a gene encoding a SPLICE erbB-2 protein. Nucleic acids isolated by screening of a cDNA or genomic DNA library can be sequenced by standard techniques.
  • An isolated and purified nucleic acid molecule of the invention which is DNA can also be isolated by selectively amplifying a nucleic acid encoding a SPLICE erbB-2 protein using the polymerase chain reaction (PCR) methods and cDNA or genomic DNA. It is possible to design synthetic oligonucleotide primers from the nucleotide sequence shown in Figure 1.
  • a nucleic acid can be amplified from cDNA or genomic DNA using these oligonucleotide primers and standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • cDNA may be prepared from mRNA, by isolating total cellular mRNA by a variety of techniques, for example, by using the guanidinium-thiocyanate extraction procedure of Chirgwin et al., Biochemistry, 18, 5294-5299 (1979). cDNA is then synthesized from the mRNA using reverse transcriptase (for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Russia, FL).
  • reverse transcriptase for example, Moloney MLV reverse transcriptase available from Gibco/BRL, Bethesda, MD, or AMV reverse transcriptase available from Seikagaku America, Inc., St. Russia, FL.
  • RNA can be isolated by cloning a cDNA encoding a SPLICE erbB-2 protein into an appropriate vector which allows for transcription of the cDNA to produce an RNA molecule which encodes a protein which exhibits SPLICE erbB-2 protein activity.
  • a cDNA can be cloned downstream of a bacteriophage promoter, (e.g. a T7 promoter) in a vector, cDNA can be transcribed in vitro with T7 polymerase, and the resultant RNA can be isolated by standard techniques.
  • a bacteriophage promoter e.g. a T7 promoter
  • a nucleic acid molecule of the invention may also be chemically synthesized using standard techniques.
  • Various methods of chemically synthesizing polydeoxynucleotides are known, including solid-phase synthesis which, like peptide synthesis, has been fully automated in commercially available DNA synthesizers (See e.g., Itakura et al. U.S. Patent No. 4,598,049; Caruthers et al. U.S. Patent No. 4,458,066; and Itakura U.S. Patent Nos. 4,401,796 and 4,373,071).
  • Determination of whether a particular nucleic acid molecule encodes a protein having SPLICE erbB-2 protein activity can be accomplished by expressing the cDNA in an appropriate host cell by standard techniques, and testing the ability of the expressed protein to transform Rat-1 fibroblasts.
  • a cDNA having the biological activity of a SPLICE erbB-2 protein so isolated can be sequenced by standard techniques, such as dideoxynucleotide chain termination or Maxam-Gilbert chemical sequencing, to determine the nucleic acid sequence and the predicted amino acid sequence of the encoded protein.
  • sequence of a nucleic acid molecule of the invention may be inverted relative to its normal presentation for transcription to produce an antisense nucleic acid molecule.
  • An antisense nucleic acid molecule may be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. II. SPLICE erbB-2 Proteins
  • the present invention relates to an isolated SPLICE erbB-2 protein having an in frame deletion of sixteen (16) amino acids, two of which are conserved cysteine residues.
  • the amino acid sequence of a SPLICE erbB-2 protein, Seq I.D. No. 2, of the present invention is shown in Figure 2.
  • the inventors have demonstrated that SPLICE erbB-2 protein is expressed in human breast tissue; in low levels in normal tissue and high levels in tumor tissue.
  • the inventors have also demonstrated that the SPLICE erbB-2 protein is constitutively tyrosine phosphorated, and that this protein is capable of transforming Rat-1 fibroblasts.
  • the proteins of the present invention include truncations of the SPLICE erbB-2 protein, and analogs, and homologs and truncations thereof as described herein.
  • Truncated proteins may have an amino group (-NH2), a hydrophobic group (for example, carbobenzoxyl, dansyl, or T-butyloxycarbonyl), an acetyl group, a 9- fluorenylmethoxy-carbonyl (PMOC) group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the amino terminal end.
  • the truncated proteins may have a carboxyl group, an amino group, a T- butyloxycarbonyl group, or a macromolecule including but not limited to lipid-fatty acid conjugates, polyethylene glycol, or carbohydrates at the carboxy terminal end.
  • Proteins of the invention may also include analogs of the SPLICE erbB-2 protein as shown in Figure 2 or truncations thereof as described herein, which may include, but are not limited to one or more amino acid substitutions, insertions, and/or deletions.
  • Amino acid substitutions may be of a conserved or non-conserved nature. conserveed amino acid substitutions involve replacing one or more amino acids of the SPLICE erbB-2 amino acid sequence with amino acids of similar charge, size, and/or hydrophobicity characteristics. When only conserved substitutions are made the resulting analog should be functionally equivalent to the SPLICE erbB-2 protein.
  • Non-conserved substitutions involve replacing one or more amino acids of the SPLICE erbB-2 amino acid sequence with one or more amino acids which possess dissimilar charge, size, and /or hydrophobicity characteristics.
  • amino acid insertions may be introduced into the SPLICE erbB-2 protein.
  • Amino acid insertions may consist of single amino acid residues or sequential amino acids ranging from 2 to 15 amino acids in length.
  • the proteins of the invention also include homologs of the SPLICE erbB-2 protein ( Figure 2) and /or truncations thereof as described herein.
  • Such homologs are proteins whose amino acid sequences are comprised of the amino acid sequences of SPLICE erbB-2 protein regions from other species that hybridize under stringent hybridization conditions with a probe used to obtain the protein.
  • the invention also contemplates isoforms of the protein of the invention.
  • An isoform contains the same number and kinds of amino acids as the protein of the invention or subtle spliced differences which alter the overall number of amino acids resulting in a slight shift in molecular weight.
  • the isoforms contemplated by the present invention are those having the same properties as a SPLICE erbB-2 protein of the invention as described herein.
  • the present invention also includes SPLICE erbB-2 protein conjugated with a selected protein, or a selectable marker protein (see below) to produce fusion proteins. Further, the present invention also includes activated or phosphorylated SPLICE erbB-2 proteins of the invention. Additionally, immunogenic portions of SPLICE erbB-2 proteins are within the scope of the invention.
  • Proteins of the invention may be prepared using recombinant DNA methods. Accordingly, the nucleic acid molecules of the present invention having a sequence which encodes a SPLICE erbB-2 protein of the invention may be incorporated in a known manner into an appropriate expression vector which ensures good expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used.
  • cosmids plasmids
  • modified viruses e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses
  • the expression vectors are "suitable for transformation of a host cell", means that the expression vectors contain a nucleic acid molecule of the invention and regulatory sequences selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid molecule. Operatively linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.
  • the invention therefore contemplates a recombinant expression vector of the invention containing a nucleic acid molecule of the invention, or a fragment thereof, and the necessary regulatory sequences for the transcription and translation of the inserted protein-sequence.
  • Suitable regulatory sequences may be derived from a variety of sources, including mammalian, bacterial, fungal, viral or insect genes (For example, see the regulatory sequences described in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Selection of appropriate regulatory sequences is dependent on the host cell chosen as discussed below, and may be readily accomplished by one of ordinary skill in the art.
  • regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector. It will also be appreciated that the necessary regulatory sequences may be supplied by the native the SPLICE erbB-2 gene and/or its flanking regions.
  • the invention further provides a recombinant expression vector comprising a DNA nucleic acid molecule of the invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression, by transcription of the DNA molecule, of an RNA molecule which is antisense to the nucleotide sequence of SEQ ID NO: 1. Regulatory sequences operatively linked to the antisense nucleic acid can be chosen which direct the continuous expression of the antisense RNA molecule in a variety of cell types, for instance a viral promoter and /or enhancer, or regulatory sequences can be chosen which direct tissue or cell type specific expression of antisense RNA.
  • SMBV Supramolecular biovectors
  • the recombinant expression vectors of the invention may also contain a selectable marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.
  • selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, ⁇ - galactosidase, chloramphenicol acetyltransferase, firefly luciferase, green fluorescent protein, alkaline phosphatase, yellow fluorescent protein, blue fluorescent protein or an irnmunoglobulin or portion thereof such as the Fc portion of an immunoglobulin preferably IgG.
  • selectable marker gene Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as ⁇ -galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance, transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest.
  • the recombinant expression vectors may also contain genes which encode a fusion moiety which provides increased expression of the recombinant protein; increased solubility of the recombinant protein; and aid in the purification of the target recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.
  • Recombinant expression vectors can be introduced into host cells to produce a transformant host cell.
  • the term "transformant host cell" is intended to include prokaryotic and eukaryotic cells which have been transformed or transfected with a recombinant expression vector of the invention.
  • nucleic acid e.g. a vector
  • Prokaryotic cells can be transformed with nucleic acid by, for example, electroporation or calcium- chloride mediated transformation.
  • Nucleic acid can be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofectin, electroporation or microinjection. Suitable methods for transforming and transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press (1989)), and other laboratory textbooks.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
  • the PKT proteins of the invention may be expressed in mammalian cells, bacterial cells such as E. coli, insect cells (using baculovirus) or yeast cells.
  • Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991).
  • Mammalian cells suitable for carrying out the present invention include, among others: Cos-7 (green monkey kidney), Saqs-2 (human osteosarcoma), PC12, NIH-3T3, MONC, SY5Y, P19, G361 (human melanoma), A549 (human lung carcinoma), SW480 (human colorectal adenocarcinoma), Raji (human Burkitt's lymphoma), MOLT-4 (human lymphoblastic leukemia), K562 (human chronic myelogenous leukemia), S3 (Hela cells), HL-60 (human promyelocytic leukemia, and breast cancer cell lines; T47Ds, MCF-7s and C127s, COS (e.g., ATCC No.
  • Cos-7 green monkey kidney
  • Saqs-2 human osteosarcoma
  • PC12 NIH-3T3, MONC
  • SY5Y human melanoma
  • A549 human lung carcinoma
  • SW480 human colorectal a
  • Suitable expression vectors for directing expression in mammalian cells generally include a promoter (e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus, retrovirus (pBabe and LXHSD), and Simian Virus 40), as well as other transcriptional and translational control sequences.
  • a promoter e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus, retrovirus (pBabe and LXHSD), and Simian Virus 40
  • Examples of mammalian expression vectors include pCDM8 (Seed, B., (1987) Nature 329:840) and pMT2PC (Kaufman et al. (1987), EMBOJ. 6:187-195).
  • bacterial host cells suitable for carrying out the present invention include E. coli, B. subtilis, Salmonella typhimurium , and various species within the genus' Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art.
  • Suitable bacterial expression vectors preferably comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication.
  • Representative promoters include the ⁇ -lactamase (penicillinase) and lactose promoter system (see Chang et al., Nature 275:615, 1978), the trp promoter (Nichols and Yanofsky, Meth in Enzymology 101:155, 1983) and the tac promoter (Russell et al., Gene 20: 231, 1982).
  • Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes.
  • Suitable expression vectors include but are not limited to bacteriophages such as lambda derivatives or plasmids such as pBR322 (see Bolivar et al., Gene 2:9S, 1977), the pUC plasmids pUC18, pUC19, pUC118, pUC119 (see Messing, Meth in Enzymology 101:20-77, 1983 and Vieira and Messing, Gene 19:259-268, 1982), and pNH8A, pNH16a, pNHl ⁇ a, and Bluescript M13 (Stratagene, La Jolla, Calif.). Typical fusion expression vectors which may be used are discussed above, e.g.
  • inducible non-fusion expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET lid (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Yeast and fungi host cells suitable for carrying out the present invention include, but are not limited to Saccharomyces cerevisae, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus.
  • yeast S. cerivisae examples include pYepSecl (Baldari. et al., (1987) Embo J. 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al, (1987) Gene 54:113-123), and pYES2 (Invitrogen Corporation, San Diego, CA).
  • promoters, terminators, and methods for introducing expression vectors of an appropriate type into plant, avian, and insect cells may also be readily accomplished.
  • the proteins of the invention may be expressed from plant cells (see Sinkar et al., J. Biosci (Bangalore) 11:47-58, 1987, which reviews the use of Agrobacteriwn rhizogenes vectors; see also Zambryski et al., Genetic Engineering, Principles and Methods, Hollaender and Setlow (eds.), Vol. VI, pp.
  • Insect cells suitable for carrying out the present invention include cells and cell lines from Bombyx or Spodotera species.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al, (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Luckow, V.A., and Summers, M.D., (1989) Virology 170:31-39) and pBAC PAK.
  • proteins of the invention may also be expressed in non-human transgenic animals such as mice, rats, rabbits, sheep and pigs (see Hammer et al. (Nature
  • the invention further provides a recombinant expression vector for the transcription and translation in invertebrate animals including, but not limited to, zebrafish, xenopus and drosophila.
  • the proteins of the invention may also be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc. 85:2149-2154) or synthesis in homogenous solution (Houbenweyl, 1987, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I and II, Thieme, Stuttgart).
  • N-terminal or C-terminal fusion proteins comprising the SPLICE erbB-2 protein of the invention conjugated with other molecules, such as proteins may be prepared by fusing, through recombinant techniques, the N-terminal or C-terminal of the protein, and the sequence of a selected protein or selectable marker protein with a desired biological function.
  • the resultant fusion proteins contain the SPLICE erbB-2 protein fused to the selected protein or marker protein as described herein.
  • proteins which may be used to prepare fusion proteins include green fluorescent protein (GFP), yellow fluorescent protein, blue fluorescent protein, alkaline phosphatase, immunoglobulins, glutathione-S-transferase (GST), haemagglutonin (HA), and truncated myc.
  • GFP green fluorescent protein
  • GST glutathione-S-transferase
  • HA haemagglutonin
  • Phosphorylation or activation SPLICE erbB-2 proteins of the invention may be ensured using, for example, the method described in Reedijk et al. The EMBO Journal 11(4):1365, 1992. III. Utility of the Nucleic Acid Molecules and Proteins of the Invention Diagnostic Uses
  • nucleic acid molecules of the invention allow those skilled in the art to construct nucleotide probes for use in the detection of nucleic acid sequences in biological materials.
  • Suitable probes include nucleic acid molecules based on nucleic acid sequences encoding at least 6 sequential amino acids from regions of the SPLICE erbB-2 protein as shown in Figure 2.
  • a nucleotide probe may be labelled with a detectable substance such as a radioactive label which provides for an adequate signal and has sufficient half-life such as 32 P, 3 H, 1 C or the like.
  • detectable substances which may be used include antigens that are recognized by a specific labelled antibody, fluorescent compounds, enzymes, antibodies specific for a labelled antigen, and luminescent compounds.
  • An appropriate label may be selected having regard to the rate of hybridization and binding of the probe to the nucleotide to be detected and the amount of nucleotide available for hybridization.
  • Labelled probes may be hybridized to nucleic acids on solid supports such as nitrocellulose filters or nylon membranes as generally described in Sambrook et al, 1989, Molecular Cloning, A Laboratory Manual (2nd ed.).
  • the nucleic acid probes may be used to detect genes, preferably in human cells, that encode the SPLICE erbB-2 protein.
  • the nucleotide probes may be useful in the diagnosis of disorders of cell transformation such as cancer.
  • the nucleotide probes may also be used as a diagnostic tool on tissue biopsies to assess the transformed state of the cell.
  • the probes may also be used in in situ hybridization of early embryos to assess both the onset and pattern of expression during development.
  • SPLICE erbB-2 proteins of the invention can be used to prepare antibodies specific for the SPLICE erbB-2 proteins that may be used to detect the protein in a biological sample.
  • Antibodies can be prepared which bind a distinct epitope in an unconserved region of the protein.
  • An unconserved region of the protein is one which does not have substantial sequence homology to other proteins.
  • Antibodies which distinguish between SPLICE erbB-2 and other erbB-2 proteins can be prepared which bind distinct epitopes of the protein.
  • polyclonal antisera or monoclonal antibodies can be made using standard methods.
  • a mammal e.g., a mouse, hamster, or rabbit
  • an immunogenic form of the peptide which elicits an antibody response in the mammal.
  • Techniques for conferring immunogenicity on a peptide include conjugation to carriers or other techniques well known in the art.
  • the protein or peptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassay procedures can be used with the immunogen as antigen to assess the levels of antibodies.
  • antisera can be obtained and, if desired, polyclonal antibodies isolated from the sera.
  • antibody producing cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
  • myeloma cells can be harvested from an immunized animal and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells.
  • Such techniques are well known in the art, (e.g., the hybridoma technique originally developed by Kohler and Milstein (Nature 256, 495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol.
  • Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the peptide and the monoclonal antibodies can be isolated. Therefore, the invention also contemplates hybridoma cells secreting monoclonal antibodies with specificity for the SPLICE erbB-2 protein as described herein.
  • antibody as used herein is intended to include fragments thereof which also specifically react with a SPLICE erbB-2 protein, or peptide thereof, having the activity of the SPLICE erbB-2 protein.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above. For example, F(ab')2 fragments can be generated by treating antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments.
  • Chimeric antibody derivatives i.e., antibody molecules that combine a non-human animal variable region and a human constant region are also contemplated within the scope of the invention.
  • Chimeric antibody molecules can include, for example, the antigen binding domain from an antibody of a mouse, rat, or other species, with human constant regions.
  • Conventional methods may be used to make chimeric antibodies containing the immunoglobulin variable region which recognizes the gene product of SPLICE erbB-2 antigens of the invention (See, for example, Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81,6851 (1985); Takeda et al., Nature 314, 452 (1985), Cabilly et al., U.S. Patent No.
  • Monoclonal or chimeric antibodies specifically reactive with a protein of the invention as described herein can be further humanized by producing human constant region chimeras, in which parts of the variable regions, particularly the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin.
  • Such immunoglobulin molecules may be made by techniques known in the art, (e.g., Teng et al, Proc. Natl. Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., Immunology Today, 4, 7279 (1983); Olsson et al, Meth. Enzymol., 92, 3-16 (1982)), and PCT Publication WO92/06193 or EP 0239400).
  • Humanized antibodies can also be commercially produced (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain.)
  • Specific antibodies, or antibody fragments, reactive against proteins of the invention may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with peptides produced from the nucleic acid molecules of the present invention.
  • complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (See for example Ward et al., Nature 341, 544-546: (1989); Huse et al., Science 246, 1275-1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)).
  • phage expression libraries See for example Ward et al., Nature 341, 544-546: (1989); Huse et al., Science 246, 1275-1281 (1989); and McCafferty et al. Nature 348, 552-554 (1990)).
  • SCID-hu mouse for example the model developed by Genpharm, can be used to produce antibodies, or fragments thereof.
  • Antibodies specifically reactive with the SPLICE erbB-2 protein, or derivatives thereof, such as enzyme conjugates or labeled derivatives, may be used to detect SPLICE erbB-2 protein in various biological materials, for example they may be used in any known immunoassays which rely on the binding interaction between an antigenic determinant of the protein, and the antibodies. Examples of such assays are radioimmunoassays, enzyme immunoassays (e.g. ELISA), immunofluorescence, immunoprecipitation, latex agglutination, hemagglutination, and histochemical tests.
  • the antibodies may be used to detect and quantify the SPLICE erbB-2 protein in a sample in order to determine its role in particular cellular events or pathological states, and to diagnose and treat such pathological states.
  • the antibodies may also be used on biopsied tissue to assess whether or not a cell is transformed.
  • antibodies of the invention may be used in immuno-histochemical analyses, for example, at the cellular and sub-subcellular level, to detect the SPLICE erbB-2 protein, to localise it to particular cells and tissues and to specific subcellular locations, and to quantitate the level of expression.
  • Cytochemical techniques known in the art for localizing antigens using light and electron microscopy may be used to detect the SPLICE erbB-2 protein.
  • an antibody of the invention may be labelled with a detectable substance and the SPLICE erbB-2 protein may be localised in tissue based upon the presence of the detectable substance.
  • detectable substances include various enzymes, fluorescent materials, luminescent materials and radioactive materials.
  • suitable enzymes include horseradish peroxidase, biotin, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate.
  • Antibodies may also be coupled to electron dense substances, such as ferritin.or colloidal gold, which are readily visualised by electron microscopy.
  • Indirect methods may also be employed in which the primary antigen-antibody reaction is amplified by the introduction of a second antibody, having specificity for the antibody reactive against a SPLICE erbB-2 protein.
  • a second antibody having specificity for the antibody reactive against a SPLICE erbB-2 protein.
  • the antibody having specificity against the protein is a rabbit IgG antibody
  • the second antibody may be goat anti-rabbit gamma-globulin labelled with a detectable substance as described herein.
  • SPLICE erbB-2 protein may be localized by autoradiography.
  • the results of autoradiography may be quantitated by determining the density of particles in the autoradiographs by various optical methods, or by counting the grains.
  • SPLICE erbB-2 protein is an erbB-2 receptor with intracellular tyrosine kinese activity which is constitutively tyrosine phosphorylated having an in frame deletion of 16 amino acids, two of which are conserved cysteine residues.
  • This novel receptor is capable of transforming Rat-1 fibroblasts and as such likely plays a role in regulating transformation of cells. Therefore, the above described methods for detecting nucleic acid molecules and SPLICE erbB-2 proteins of the invention, can be used to monitor cell transformation.
  • SPLICE erbB-2 protein of the invention is likely involved in the regulation of cell signalling pathways that control cell transformation.
  • the present invention provides a method of modulating cell transformation comprising administering an effective amount of a SPLICE erbB-2 protein or a nucleic acid encoding a SPLICE erbB-2 protein to a cell or animal in need thereof.
  • effective amount means an amount effective, at dosages and for periods of time necessary to achieve the desired results.
  • the present invention provides a method of modulating tumor cell proliferation.
  • the invention provides a method of inhibiting or reducing tumor cell proliferation by administering an agent that inhibits the expression or the biological activity of the SPLICE erbB-2 protein.
  • Agents that inhibit the activity of a SPLICE erbB-2 protein include antibodies to the protein.
  • Agents that inhibit the expression of the SPLICE erbB-2 gene include antisense oligonucleotides to a SPLICE erbB-2 nucleic acid sequence. Both of these are described above.
  • the invention also provides methods for identifying substances which are capable of binding to a SPLICE erbB-2 protein. In particular, such methods may be used to identify substances capable of binding to, and in some cases activating (i.e., phosphorylating), or deactivating the protein of the invention.
  • Substances which can bind with a SPLICE erbB-2 protein of the invention may be identified by reacting the protein with a substance which potentially binds to the protein, and assaying for complexes, for free substance, or for non-complexed SPLICE erbB-2 protein, or for activation or deactivation (as the case may be) of the protein.
  • a yeast two hybrid assay system may be used to identify proteins which interact with the SPLICE ERBB- 2 protein (Fields, S. and Song, O., 1989, Nature, 340:245-247).
  • Conditions which permit the formation of a substance and SPLICE erbB-2 protein complexes may be selected having regard to factors such as the nature and amounts of the substance and the protein.
  • the substance-protein complex, free substance or non-complexed proteins may be isolated by conventional isolation techniques, for example, salting out, chromatography, electrophoresis, gel filtration, fractionation, absorption, polyacrylamide gel electrophoresis, agglutination, or combinations thereof.
  • antibody against the SPLICE erbB-2 protein or the substance, or labelled SPLICE erbB-2 protein, or a labelled substance may be utilized.
  • the antibodies, proteins, or substances may be labelled with a detectable substance as described above.
  • Substances which bind to and activate or deactivate SPLICE erbB-2 protein of the invention may be identified by assaying for phosphorylation of the tyrosine residues of the protein.
  • SPLICE erbB-2 protein or the substance used in the method of the invention may be immobilized.
  • the SPLICE erbB-2 protein or substance may be bound to a suitable carrier.
  • suitable carriers are agarose, cellulose, dextran, Sephadex, Sepharose, carboxymethyl cellulose polystyrene, filter paper, ion-exchange resin, plastic film, plastic tube, glass beads, polyamine-methyl vinyl-ether-maleic acid copolymer, amino acid copolymer, ethylene-maleic acid copolymer, nylon, silk, etc.
  • the carrier may be in the shape of, for example, a tube, test plate, beads, disc, sphere etc.
  • the immobilized protein or substance may be prepared by reacting the material with a suitable insoluble carrier using known chemical or physical methods, for example, cyanogen bromide coupling.
  • proteins or substance may also be expressed on the surface of a cell using the methods described herein.
  • the invention also contemplates a method for assaying for an agonist or antagonist of the binding of the SPLICE erbB-2 protein with a substance which is capable of binding with the SPLICE erbB-2 protein.
  • the agonist or antagonist may be an endogenous physiological substance or it may be a natural or synthetic substance.
  • Substances which are capable of binding with the SPLICE erbB-2 protein may be identified using the methods set forth herein.
  • the agonists and antagonists that can be assayed using the methods of the invention may act on one or more of the binding sites on the protein or substance including agonist binding sites, competitive antagonist binding sites, non-competitive antagonist binding sites or allosteric sites.
  • the invention also makes it possible to screen for antagonists that inhibit the effects of an agonist of the interaction of the SPLICE erbB-2 protein with a substance which is capable of binding to the protein.
  • the invention may be used to assay for a substance that competes for the same binding site of the SPLICE erbB-2 protein.
  • the methods described above may be used to identify a substance which is capable of binding to an activated SPLICE erbB-2 protein, and to assay for an agonist or antagonist of the binding of activated protein, with a substance which is capable of binding with activated SPLICE erbB-2 protein.
  • An activated (i.e., phosphorylated) SPLICE erbB-2 protein may be prepared using the methods described (for example in Reedijk et al. The EMBO Journal, 11(4):1365, 1992) for producing a constitutively tyrosine phosphorylated protein.
  • intracellular substances which are capable of binding to SPLICE erbB-2 protein may be identified using the methods described herein.
  • the invention further provides a method for assaying for a substance that affects an SPLICE erbB-2 protein regulatory pathway comprising administering to a non-human animal or to a tissue of an animal, a substance suspected of affecting a SPLICE erbB-2 protein regulatory pathway, and quantitating the protein or nucleic acids encoding the protein, or examining the pattern and /or level of expression of SPLICE erbB-2 protein, in the non-human animal or tissue.
  • SPLICE erbB-2 protein may be quantitated and its expression may be examined using the methods described herein.
  • Substances identified by the methods described herein may be used for modulating SPLICE erbB-2 protein regulatory pathways and accordingly may be used in the treatment of conditions involving perturbation of SPLICE erbB-2 protein signalling pathways.
  • the substances may be particularly useful in the treatment of disorders of cell proliferation such as cancer.
  • SPLICE erbB-2 protein is involved in cell transformation and inhibitors of this protein may be useful in reversing disorders involving cell transformation such as cancer.
  • stimulators of the SPLICE erbB-2 protein may be useful in the treatment of disorders requiring stimulation of proliferation.
  • substances that stimulate SPLICE erbB-2 protein may be used to stimulate cell proliferation.
  • Substances which stimulate cell proliferation may be useful in the treatment of conditions involving damaged cells including conditions in which degeneration of tissue occurs such as arthropathy, bone resorption, inflammatory disease, degenerative disorders of the central nervous system and for promoting wound healing.
  • All of the above described substances may be formulated into pharmaceutical compositions for adminstration to subjects in a biologically compatible form suitable for administration in vivo.
  • biologically compatible form suitable for administration in vivo is meant a form of the substance to be administered in which any toxic effects are outweighed by the therapeutic effects.
  • the substances may be administered to living organisms including humans, and animals. In this context and throughout this specification animal means any member of the animal kingdom.
  • a therapeutically active amount of pharmaceutical compositions of the present invention is defined as an amount effective, at dosages and for periods of time necessary to achieve the desired result.
  • a therapeutically active amount of a substance may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of antibody to elicit a desired response in the individual. Dosage procedures may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
  • Active substance may be administered in a convenient manner such as by injection (subcutaneous, intravenous, etc.), oral administration, inhalation, transdermal application, or rectal administration. Depending on the route of administration, the active substance may be coated in a material to protect the compound from the action of enzymes, acids and other natural conditions which may inactivate the compound. If the active substance is a nucleic acid encoding a SPLICE erbB-2 protein or an antisense oligonucleotide it may be delivered using techniques known in the art. Recombinant molecules comprising an antisense sequence or oligonucleotide may be directly introduced into cells or tissues in vivo using delivery vehicles such as retroviral vectors, adeno viral vectors and DNA virus vectors. They may also be introduced using physical techniques such as microinjection and electroporation or chemical methods such as co-precipitation and incorporation of DNA into liposomes.
  • compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle.
  • Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985).
  • the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
  • Reagents suitable for applying methods of the invention to identify substances that affect SPLICE erbB-2 protein may be packaged into convenient kits providing the necessary materials packaged into suitable containers.
  • the kits may also include suitable supports useful in performing the methods of the invention.
  • the invention also provides methods for studying the function of SPLICE erbB-2 protein.
  • Cells, tissues and non-human animals that express or over-express SPLICE erbB-2 protein may be prepared by transfecting cells, tissues or oocytes (to prepare transgenic animals) with a recombinant expression vector of the invention (as described previously).
  • transgenic technology via nuclear oocyte microinjection of naked DNA
  • Cells, tissues, and non-human animals lacking in SPLICE erbB-2 protein expression or partially lacking in SPLICE erbB-2 protein expression may be developed using recombinant expression vectors of the invention having specific deletion or insertion mutations in the SPLICE erbB-2 gene.
  • a recombinant expression vector may be used to inactivate or alter the endogenous gene by homologous recombination, and thereby create a SPLICE erbB-2 protein deficient cell, tissue or animal.
  • Null alleles may be generated in cells, such as embryonic stem cells by deletion mutation.
  • a recombinant SPLICE erbB-2 gene may also be engineered to contain an insertion mutation which inactivates the SPLICE erbB-2 protein.
  • Such a construct may then be introduced into a cell, such as an embryonic stem cell, by a technique such as transfection, electroporation, injection etc.
  • Cells lacking an intact SPLICE erbB-2 gene may then be identified, for example by Southern blotting, Northern Blotting or by assaying for SPLICE erbB-2 protein using the methods described herein.
  • Such cells may then be fused to embryonic stem cells to generate transgenic non-human animals deficient in SPLICE erbB-2 protein.
  • Germline transmission of the mutation may be achieved, for example, by aggregating the embryonic stem cells with early stage embryos, such as 8 cell embryos, in vitro; transferring the resulting blastocysts into recipient females and; generating germline transmission of the resulting aggregation chimeras.
  • Such a mutant animal may be used to define specific cell populations, developmental patterns and in vivo processes, normally dependent on SPLICE erbB-2 protein expression.
  • the present invention also includes the preparation of tissue specific knock-outs of the SPLICE erbB-2 gene.
  • RT-PCR of total RNA from Normal and Tumor Breast Tissue Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)
  • RNA isolated from a human prostate carcinoma cell line (DU145) as previously described (Siegel et al, 1994). After incubation at 37°C for 2 h, 1.0 ⁇ l of the RT mix was added to the following: 0.5 ⁇ l of each oligonucleotide primer (lO ⁇ M), 2.0 ⁇ l of 10X PCR buffer (200 mM Tris-HCl [pH 8.4], 500 mM KC1), 1.0 ⁇ l of 50 mM MgCl 2 , 1.0 ⁇ l of deoxynucleotide triphosphate (dNTP) mix (lOmM
  • PCR amplification was performed for 30 cycles of 1 min at 94°C, 30 s at 55 °C, and 1 min at 72°C.
  • Both the wild-type and alternately spliced form of erbB-2 wefe amplified with the following primers; AB12904 ( T TT C C A GA T GA GGA GGGC ) ( S E Q . I . D . N O . 3) and A B 1 2903 (CGGAATTCCTGTCACCAGCTGCACCGT) (SEQ. I.D. No. 4), which amplify the region corresponding to nucleotides 1996-2544 of the human erbB-2 cDNA (Coussens et ⁇ Z.,1985).
  • Rat-1 fibroblasts were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin, streptomycin, and fungizone (GIBCO BRL). Stable cell lines were derived by electroporation as described previously (Siegel et al, 1994). DNA was introduced into the Rat-1 cells at a 50:1 ratio of expression plasmid to puromycin resistance plasmid (PGK-puro) and resistant colonies were selected for 10 days in media containing puromycin (3.0 ⁇ g/ml) prior to deriving clonal cell lines.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • FBS fetal bovine serum
  • streptomycin penicillin
  • streptomycin fungizone
  • the erbB-2 focus assays were performed essentially as described (Siegel et al, 1994) with the exception that monolayers were maintained for 12 days in DMEM supplemented with 2% FBS, penicillin, streptomycin, and fungizone. Five ⁇ g of each pJ4 ⁇ expression plasmid was electroporated per lxlO 6 cells.
  • expression plasmids bearing the wild- type (WT) erbB-2 cDNA, a transforming erbB-2 allele possessing the point mutation in the transmembrane domain (VE), a previously characterized sporadic deletion discovered in the neu proto-oncogene (Siegel et al , 1994) (ECD DEL) or the spliced form of erbB-2 (SPLICE) were electroporated into Rat-1 cells and assessed for their capacity to induce transformed foci (Figure 4a).
  • TNE lysis buffer 50 mM Tris [pH 8.0], 150 mM NaCl, 1% Nonidet P-40, 2 mM EDTA [pH 8.0], 1 mM sodium orthovanadate, 10 mM sodium fluoride, 10 ⁇ g/ml Aprotinin, 10 ⁇ g/ml Leupeptin). Lysates were cleared by centrifugation for 10 min at 4°C and the protein concentration determined by Bradford assay (Bio-Rad).
  • ErbB-2 was immunoprecipitated (Ab-5; 1 ⁇ g/600 ⁇ g total protein) and blotted (Ab-3; 1:1000) using mouse monoclonal antibodies purchased from Oncogene Research Products, Inc. Phosphotyrosine blots were probed with mouse anti-phosphotyrosine antibodies (PY20; 1:1000) obtained from Transduction Laboratories. Horseradish peroxidase conjugated anti-mouse secondary antibodies (1:2500) (Jackson Laboratories) were used in each case.
  • Rat-1 cells expressing various erbB-2 alleles were generated.
  • protein extracts derived from the various cell lines were immunoprecipitated with erbB-2 specific antibodies and subjected to immunoblot analyses with anti-phosphotyrosine antibodies.
  • tyrosine phosphorylated erbB-2 could be easily detected in transformed cells expressing the oncogenic alleles of erbB-2 but was barely detectable in morphologically normal cell lines expressing elevated levels of wild-type erbB-2.
  • deletions in the EGFR (Sugawa et al., 1990; Wong et al., 1992) and missense mutations in RET (Mulligan et aL, 1993) which also affect cysteine residues situated in the extracellular domains of these RTKs, have frequently been observed in human gliomas and endocrine neoplasias, respectively.
  • the observed mutations serve to activate the transforming ability of these receptors (Nishikawa et aL, 1994; Santoro et al., 1995; Asai et aL, 1995).
  • Therapeutic reagents specific to the receptor encoded by this spliced form of erbB-2 may have important diagnostic and therapeutic implications in the treatment of human breast cancer.
  • RT-PCR Reverse-transcriptase polymerase chain reaction
  • ErbB-2 represents the wild-type receptor
  • ErbB-2 VE
  • ECD DEL ErbB-2
  • SPLICE ErbB-2 contains the deletion encoded by the alternatively spliced erbB-2 message.
  • a portion of the transmembrane domain is indicated by the open box and designated TM.
  • Altered ErbB-2 receptors are constitutively tyrosine phosphorylated when expressed in Pat-1 cells
  • Lysates from stable cell lines expressing the various ErbB-2 receptors were immunoprecipitated for ErbB-2 (Ab5, Oncogene Research Products) and the immunoprecipitates were subjected to immunoblot analysis with ErbB-2/Neu-specific antibodies (AW, Oncogene Research Products).
  • Ratl indicates the parental cell line which does not contain any of the erbB-2 expression plasmids.
  • the position of ErbB-2 is indicated in each panel by the arrow.
  • RON transcript induces constitutive tyrosine kinase activity and an invasive phenotype.
  • RETas Activation of RETas a dominant transforming gene by germline mutations of MEN2A and MEN213. Science 267:381- 383. 10. Siegel, P. M., D. L. Dankort, W. R. Hardy and W. J. Muller. 1994. Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors. Mol. Cell. Biol. 14:7068-7077.
  • Rat-1 fibroblasts Three independent focus assays were performed with established Rat-1 fibroblasts. The first two experiments were conducted with plasmid DNA from the same large-scale preparation, while the third experiment was performed with DNA obtained from an independent plasmid preparation.
  • the Rat-1 fibroblasts were maintained in a monolayer for 14 days following electroporation at which time they were stained with Geimsa.
  • b Values listed represent the mean number of foci counted on 8 plates 1 the standard deviations.
  • c Values represent the ratio of the mean number of foci obtained for each constuct with respect to pJ4 ⁇ B2(VE).
  • d Values listed represent the mean transforming abilities from all three experiments 1 the standard deviations.

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Abstract

Cette invention se rapporte à une nouvelle forme épissée de l'oncogène Erb-2 qui code un récepteur d'Erb-2 à phosphorylation constitutive par la tyrosine. Cette protéine présente une délétion dans le cadre de16 acides aminés, dont deux sont des restes systéine conservés. Cette protéine peut servir à mettre au point des remèdes pour limiter la transformation cellulaire, telle qu'elle se produit notamment dans le cancer du sein.
PCT/CA1999/000912 1998-10-02 1999-10-01 Forme epissee de l'oncogene erbb-2/neu WO2000020579A1 (fr)

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