WO1991013173A1 - METHODES DE DIAGNOSTIQUE UTILISANT DES GENES PTPase ET LEURS UTILISATIONS - Google Patents

METHODES DE DIAGNOSTIQUE UTILISANT DES GENES PTPase ET LEURS UTILISATIONS Download PDF

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WO1991013173A1
WO1991013173A1 PCT/US1991/001432 US9101432W WO9113173A1 WO 1991013173 A1 WO1991013173 A1 WO 1991013173A1 US 9101432 W US9101432 W US 9101432W WO 9113173 A1 WO9113173 A1 WO 9113173A1
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ptpase
gene
probe
protein
ptpib
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PCT/US1991/001432
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Arthur M. Bruskin
David E. Hill
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Applied Biotechnology, Incorporated
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Priority to CA002054725A priority Critical patent/CA2054725A1/fr
Publication of WO1991013173A1 publication Critical patent/WO1991013173A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57426—Specifically defined cancers leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/914—Hydrolases (3)
    • G01N2333/916—Hydrolases (3) acting on ester bonds (3.1), e.g. phosphatases (3.1.3), phospholipases C or phospholipases D (3.1.4)

Definitions

  • the present invention is directed to the use of vectors, transformants and cell lines containing a supressor gene and the use of such gene for expression, diagnostic and therapeutic means. More particularly, the invention is directed to the use of PTPase genes, preferably PTPIB, for diagnostic and therapeutic uses.
  • Vanadate a potent inhibitor of PTPases, causes a transient increase in the cellular phosphotyrosine content as well as a phenotypic transformation of some fibroblast cell lines [Klarlund, J.K. ,
  • Protein phosphotyrosyl phosphatases have been identified in many different eukaryotic cell types in a broad range of sizes from both particulate and soluble cell fractions [Jones, S.W., et al, J. Biol. Che ⁇ t. 264:7747-7753: Lau, K.H. . , et al, Biochem. J.
  • PTPases comprise a diverse family of proteins consisting of both transmembrane glycoproteins and cytosolic proteins. While the enzymology of intracellular PTPases is being studied [Lau, K.H. , et al., Biochem. J. 257:23-36 (1989); Tonks, N.K. , et al, Adv. Prot. Phosnhatases 1:149-180 (1989)], physiologically relevant substrates for these enzymes have not yet been identified, nor has the mode of regulation of enzymatic activity been elucidated.
  • PTPase IB from placenta and PTPase 5 from bovine brain have been reported to dephosphorylate the phosphotyrosyl forms of insulin receptor and v-src. respectively, in vitro [Jones, S.W., et al, J. Biol. Chem. 264:7747-7753 (1989); Tonks, N.K. , et al, J. Biol. Chem. 26_3:6731-6737 (1988)] and purified PTPase IB injected into Xenopus oocytes delays insulin-dependent maturation [Tonks, N.K. , et al, Adv. Prot. Phosphatases 5:149-180 (1989)].
  • Malignant transformation is believed to result from the cumulative effects of multiple genetic lesions.
  • Two classes of genetic lesions have been defined. The best understood class is the dominant, activating mutations that generate oncogenes [Bishop, J.M. , Science 215:305-311 (1987)].
  • a second class is the loss of function mutations that lead to neoplastic growth. Such mutations are associated with growth suppressor genes [Hansen, M.F., et al. , Cell 53:172-173 (1988); Weinberg, R. , Biochemistry 28:8263-8269 (1989) which are incorporated by reference] , and are sometimes referred to as tumor suppressor genes or anti-oncogenes.
  • neoplastic growth cancer It would be desirable to have a method of diagnosis for the presence of neoplastic growth (cancer) based upon identifying the absence of a functional portion of a suppressor gene or an alteration in the sequence of such gene and/or by the reduced activity of the protein product of the suppressor gene.
  • ⁇ It would also be useful to have a means of counteracting this neoplastic growth by being able to supply a therapeutic amount of the suppressor gene product to counteract the neoplastic growth caused by its loss or alteration.
  • the gene is a member of the PTPase gene family, such as PTPIB, LAR, CD45, PTPase 5, or T-cell PTPase. More preferably, the gene is a PTPIB gene.
  • the human PTPIB gene is located on the long arm of chromosome 20, and localizes near the interface of bands ql3.1 and ql3.2; referred to as localizing between 20 ql3.1 - 13.2.
  • the presence of malignancy in humans can be determined by assaying for a deletion of a functional portion or alteration of a suppressor gene such as a PTPase gene, preferably, the PTPIB gene. This can be accomplished by the use of a nucleic acid probe based on the nucleotide sequence of the suppressor gene or by the use of an antibody probe to the gene's expression product.
  • an alteration in the suppressor gene can be determined by comparing a preselected cell from an individual with a normal cell from that individual.
  • the preselected cell is compared against a predetermined base line. Preferably, one compares the level of the suppressor gene product to a "normal" base level, with a low level being indicative of a neoplastic condition.
  • the suppressor gene is used to diagnose the presence of a cancer or precancerous condition, preferably for the presence of a cancer such as leukemia. More preferably, acute nonlymphocytic leukemia (ANLL) .
  • a cancer such as leukemia. More preferably, acute nonlymphocytic leukemia (ANLL) .
  • ANLL acute nonlymphocytic leukemia
  • Preferred precancerous conditions diagnosed are myelodysplastic syndrome (MDS) such as idiopathic acquired sideroblastic anemia or refractory anemia with excess blasts, as well as myeloproliferative disorders (MPDs) such as polycythemia vera.
  • MDS myelodysplastic syndrome
  • MPDs myeloproliferative disorders
  • Figure 1 is a schematic of the nucleotide and predicted amino acid sequence for PTPIB cDNA.
  • Figure 2A is a schematic of the primary translation product of the cDNA of PTPase IB and T-cell PTPase.
  • Figure 2B is the amino acid alignment of the deduced carboxy termini of PTPase IB and T-cell PTPase.
  • Figure 3 is a picture of an autoradiograph of the _n vitro translation product of the PTPIB cDNA.
  • Figure 4A is a schematic representation of the PTPIB cDNA and the genomic PTPIB DNA.
  • Figure 4B is a representation of the DNA sequences of the intron/exon and exon/intron junctions for the 5 exons (A-E) identified in Figure 4A.
  • Figures 5A-C are fluorescence in situ hybriderization localization of the PTPIB gene.
  • Figure 5D is a schematic representation of human chromosome 20 with a summary of the PTPIB gene placement.
  • Figures 6A-F are pictures of NIH 3T3 cells and 3T3 3-18 cells.
  • Figure 7 is a schematic of a plasmid containing the PTPIB cDNA.
  • Figure 8 is a schematic of a clone containing the PTPIB genome.
  • Figures 9 is gels of PTPase IB.
  • Figure 10 is a Western analysis of PTPIB monoclonal antibodies CE5-1K and AE4-2J.
  • Figure 11 is a picture of an autoradiograph of immunoprecipitation of 3T3 and 3-18 cells with PTPIB monoclonal antibodies.
  • Figure 12 is a Western analysis of PTPase expressed from vaccinia infected cells and PTPIB monoclonal antibodies.
  • Figure 13 is a Western analysis of tumor cells from nude mice that were injected with neu TM infected cells.
  • Figure 14 is a Western analysis of 3T3, 3-18 and 43-5, a 3T3 cell line transfected with the PTPIB gene under the control of an inducible promoter.
  • Figure 15 is a picture of an autoradiogram of an in vitro tyrosine kinase assay.
  • Figure 16 is a schematic of pAbT4587.
  • Figure 17A is a schematic of pAbT9110 and Figure 17B is a schematic of pAbT9112.
  • Figure 18 is a schematic of an inducible PTPase expression plasmid.
  • PTPase genes are associated with neoplastic growth.
  • One mechanism for generating loss of function mutations is deletion of a putative growth suppressor gene which may be accompanied by observable chromosomal abnormalities.
  • Two growth suppressor genes have been Isolated based on this criteria [Friend, S.H., et al, Nature 128:643-647 (1986); Fung, Y.K.T., et al, Science 236:1657-1661 (1987); Lee, W.H. , et al, Science 235:1394-1399 (1987); Fearon, E.R. , et al, Science 247:49-56 (1990) which are incorporated by reference] .
  • the probe can be a nucleotide probe or an antibody probe.
  • the presence of malignancy can be determined by assaying for the deletion of a functional portion or alteration of a suppressor gene.
  • the suppressor gene is a member of the PTPase gene family, e.g., PTPIB (sometimes referred to as PTPase IB), T-cell PTPase, LAR, CD45 and PTPase 5.
  • Figure 1 shows the nucleotide sequence and predicted amino acid sequence for the PTPIB cDNA.
  • the cDNA sequence of the PT._3 gene has a 1305 nucleotide open reading frame, which predicts a protein of 435 amino acids having a molecular weight of about 49,966 (in Figure 1 the termination codon is marked by an X).
  • Nucleotide numbering in Figure 1 is on the right with the sequence numbered from the first ATG codon observed in the single open reading frame.
  • the underlined amino acid sequences correspond to the oligopeptides chosen for construction of the degenerate oligonucleotides used to screen the cDNA library) .
  • the Asn321 residue marking the carboxy-terminus of the purified 37000 dalton PTPase IB is underlined. A putative poly(A)addition site is observed at nucleotides 3123-3128.
  • FIG. 2 is a schematic representation of the amino acid alignments of PTPase IB and T cell PTPase.
  • Figure 2A shows the primary translation product of each cDNA represented by the individual lines. The filled boxes connecting the two lines indicates oligopeptides of seven or more amino acids that are identical between the two proteins. The numbers above the lines refer to PTPase IB amino acid numbering.
  • the cross-hatched box represents the hydrophobic region at the carboxy-terminus of the two proteins.
  • Figure 2B shows the alignment of the deduced carboxy-termini of PTPase IB (labelled PTP IB) and T-Cell PTPase (labelled PTP TC) . Amino acids are shown in one-letter code, and the amino acid identities between the two regions are indicated. The hydrophobic region containing 8 out of 19 identical amino acids is boxed.
  • the cDNA sequence can be used in preparing nucleotide probes for detection of deletions and alterations in the suppressor gene.
  • the genomic sequence is preferably used to determine the chromosomal location.
  • Figure 4 is a schematic representation of the genomic structure and cDNA structure for the PTPIB gene with exon/intron structure of this gene shown. Southern analysis of the genomic DNA identified single EcoRI, BamHI and Hindlll fragments when the coding region of the cDNA was used as a probe. However, since a single Hindlll site is present in the cDNA sequence, there may be an additional smaller genomic Hindlll fragment not seen on the Southern blots.
  • Figure 4A is a schematic representation of the cDNA clone with the open box representing the 1305 nucleotide open reading frame, upper figure, and the 13 kb genomic fragment, lower figure shown. The genomic figure is not drawn to scale. Exons, identified by determining exon/intron junctions through comparison of cDNA and genomic DNA sequences, are shown as open boxes labelled A through E and are mapped onto the cDNA. Various restriction endonuclease sites are shown for both clones (Bm - BamHI; R - EcoRI; H - Hindlll; Q - Sad; S - Sphl).
  • Figure 4B is the DNA sequences of the intron/exon and exon/intron junctions for the five exons (A-E) identified in Figure 4A.
  • Intron sequences are in lower case letter ; exon sequences are in upper case letters. Numbers above the exon sequences correspond to the cDNA numbering in Figure 1.
  • Consensus nucleotides for intron sequences [Shapiro, M.B.,et al, Nucl. Acids Res. 15. 7155-7174 (1987)] are underlined. The reading frame and translation products are shown beneath the exon sequences.
  • Exon E has only an intron-exon junction because it contains the stop codon and the 3' untranslated region.
  • a functional portion of the gene is a sequence the deletion of which negatively affects the growth suppressing activity of the resultant gene product. Alterations of the gene can be distinguished from allelic variation by comparing the PTPase gene or gene product in the cell being tested with a PTPase gene or gene product from a "normal" cell (i.e. one not believed to be malignant) from the same individual.
  • a deletion of a functional unit can be any number of nucleotides that result in an altered or inactive gene product.
  • the deletion is preferably at least about 100 nucleotides, more preferably at least about 250 nucleotides, still more preferably at least about 400 nucleotides.
  • the deletion is at least about one exon of the gene.
  • the PTPIB gene it preferably is a deletion of at least about 250 nucleotides, more preferably at least about 400 nucleotides, still more preferably, at least about 600 nucleotides.
  • a deletion of at least most of the nucleotides, preferably all the nucleotides, of one of the five exons, for example, exon A, is also preferred for detecting the presence of cancer.
  • the nucleotide probe corresponds to at least about 20 nucleotides of a human PTPase gene. More preferably, it corresponds to at least about 25% of the cDNA of a PTPase gene, still more preferably, it corresponds to at least about 50% of the cDNA of PTPase gene, even more preferably it corresponds to at least about 70% of the cDNA of a PTPase gene.
  • the nucleotide probe corresponding to at least about 25% of the coding sequence of a PTPase gene, more preferably, at least about 50% of the coding sequence of a PTPase gene, still more preferably, at least about 70% of the coding sequence of a PTPase gene.
  • the PTPase gene is the PTPIB gene.
  • the coding sequence refers to either the cDNA or the genomic sequences (i.e. the exons and the intervening sequences), but the probe preferably is based upon the cDNA sequences.
  • nucleotide probe When using a nucleotide probe, one can determine whether there is a deletion in a functional portion of the gene by a variety of techniques well known in the art such as Southern blot analysis [Southern, E.M. , J. Mol. Biol. 98:503 (1975)] or in situ hybridization [Lawrence, J.B., Cell 52:51 (1988)].
  • the probe can be labelled, preferably with a radioactive isotope, enzyme, fluorogenic, chemiluminescent or electro-chemical material.
  • a probe corresponding to a genomic sequence is labelled, preferably with a radioactive isotope, enzyme, fluorogenic, chemiluminescent or electro-chemical material.
  • one uses a nucleotide probe to compare the nucleotide sequence of the test cell (i.e. predetermined cell sample) with a "normal" cell from the individual to be tested.
  • a nucleotide probe to compare the nucleotide sequence of the test cell (i.e. predetermined cell sample) with a "normal" cell from the individual to be tested.
  • predetermined cell sample is the cell sample you wish to test for malignancy. This can be from tissue, plasma, etc.
  • the alteration or deletion of a functional portion of the suppressor gene, preferably PTPase suppressor gene can indirectly be determined by the use of antibody probes using standard techniques.
  • the gene is a PTPase gene it is preferably used for the diagnosis of cancer of precancerous condition. More preferably, the diagnosis of a cancer, preferably leukemia such as acute nonlymphocytic leukemia (ANLL) .
  • ANLL acute nonlymphocytic leukemia
  • MPDs myeloproliferative disorders
  • PTPIB gene is preferred.
  • a cocktail containing probes to various functional portions of the suppressor (cDNA) gene as well as to the suppressor (genomic) gene.
  • the PTPIB suppressor gene maps as a single gene copy on the long arm of chromosome 20 in the region ql3.1-ql3.2.
  • localization of the gene to a chromosome can be determined by non-isotopic, flouresence detection of in situ hybridization with a probe based on the genomic suppressor gene.
  • Chromosome banding analysis can be performed by a variety of means well known in the art. For example, one can use
  • Figure 5 which illustrates the localization of the PTPIB gene to a single chromosome locus.
  • hybridization can be unambigously visualized in one cell by the identical labelling of both sister chromatids on each of the two homologous chromosomes.
  • Figure 5A shows DAPI-staining of metaphase chromosomes from normal human lymphocytes. The bands are sufficient to allow identification of individual chromosomes.
  • the large arrow at the top of the figure points to a pair of chromosome 20s.
  • the small arrows indicate chromosome 19, which is distinguished by a more prominent centromere and paler arms.
  • Figure 5B shows fluorescein avidin detection of hybridization with the biotin-labelled PTPIB genomic DNA probe. Both homologs of chromosome 20 label on both sister chromatids and no other chromosomal sites are labeled. The banding analysis demonstrated that this was chromosome 20 and resulted in a regional localization of the signal to the region ql3.1-ql3.2.
  • Figure 5C illustrates the position of the chromosome signal on several enlarged chromosomes at a higher magnification showing alignment of the total chromosome DAPI-fluorescence on the left of each pair with the fluorescein avidin hybridization signals on the right.
  • Figure 5D is a schematic representation of chromosome 20 with a summary of the PTPIB gene placement based on the analysis from 10 DAPI-banded chromosomes scored independently.
  • the PTPIB gene localizes near the interface of bands ql3.1 and ql3.2.
  • localization of the PTPIB gene by measurement along the length of the chromosome based on the average of 27 determinations maps the gene in the middle of ql3.1.
  • This nucleotide segment is 3143 nucleotides and has an observed 1305 nucleotide open reading frame which results in a predicted protein of 435 amino acids.
  • This nucleotide segment or a functional portion thereof can be used to generate a clone capable of expressing the PTPIB gene product by standard techniques.
  • a vector containing a PTPase gene cDNA sequence, preferably, the PTPIB gene cDNA, polyadenylation sequences downstream 3' from the sequence and preferably also containing a replication origin can be used to transfect a cell.
  • the vector will also contain a promoter to permit expression of the suppressor gene. Promoters used in the vector can be any of the known promoters and the choice is governed by the host cell one wishes to use to thereby permit expression of the desired product in the host cell of choice. Retroviral promoters are preferred. Such promoters include retroviral promoters such as Akv, SL3-3 and Friend viruses.
  • the vector can preferably also contain an enhancer, such enhancers are well known in the art, for example, viral enhancer. More preferably, it contains an enhancer which is tissue specific.
  • the vector also contains a marker gene to aid in detection of transformed cells.
  • markers are well known in the art and can include an antibiotic resistance gene such as the bacterial neomycin resistance gene, which confers a dominant selectable resistance to the antibiotic G418 in eukaryotic cells.
  • a vector such as one corresponding to a defective virus or benign virus, e.g., recombinant vaccinia virus, can be used.
  • different cell lines can differ in their ability to take up and express the transfected suppressor gene DNA by use of appropriate promoters, a wide range of host cells can be used.
  • NIH3T3, CHO, COS, SF-9.ATCC No. CRL 1711) and SF-21 are preferred.
  • mammalian cells would be preferred, but the cell lines of the present invention are not limited thereto.
  • the vector can be used to transform cell lines, for example, by being introduced into psi/2 (ecotropic) and psiAM (amphotr ⁇ pic) cell lines by a variety of methods well known to the art.
  • the preferred method is the calcium phosphate co-precipitation method [See Wigler, et al, Cell 16:777-785 (1979), which is incorporated by reference].
  • These cell lines can constitutively produce infectious, replication defective murine leukemia viruses containing a genome derived from the vector. [Cone, et al, PNAS 81:6349-6353 (1984); Mann, et al, Cell 33:153-159 (1983)].
  • Two days following transfection cells can be selected by looking for the marker, i.e. antibiotic resistance gene, which the vector would also preferably contain.
  • Antibiotic resistant clones, e.g. G418, would be evident in a short period of time, typically 7-10 days.
  • the antibiotic resistant psi/2 and psi/AM clones are isolated and clones producing large amounts of the suppressor gene product are selected. These cells are then cultured in a standard medium and harvested as needed.
  • the protein may be expressed as insoluble inclusion bodies.
  • inclusion bodies can be isolated by standard means, e.g., centrifugation of cell lysates. Thereafter, the protein can be readily isolated by standard Isolation techniques.
  • the protein produced from these cells can then be used to generate an antibody to the suppressor gene product (protein) .
  • the antibody generated can be polyclonal or monoclonal depending upon the particular application for which it is designed.
  • Such antibodies can be prepared by techniques well know to the skilled artisan.
  • the protein or an antigenic portion thereof can be conjugated to keyhole limpet hemocyanin (KLH) and used to raise an antibody in an animal such as a rabbit.
  • KLH keyhole limpet hemocyanin
  • the peptide-KLH conjugate Is injected several times over a period of about two months to generate antibodies.
  • the antibody Is then collected from serum by standard techniques.
  • monoclonal antibodies can be produced in cells which produce antibodies to the proteins by using standard fusion techniques for forming hybridoma cells.
  • hybridomas can be generated by immunization of mice with viable cells transformed by the suppressor gene-containing vector, which express the suppressor gene product.
  • these cells express the full length protein such as the full length PTPIB gene product shown in Figure 1.
  • the full length protein as the immunogen, it is possible to generate a collection of monoclonal antibodies with specificities that span the entire length of the protein. This is as opposed to the use of peptide im unogens, or short polypeptides generated by prokaryotic systems, which present a more limited number of epitopes from the original protein and hence, raise an immune response of more limited specificity, such as would be the case with the Charbonneau protein (Proc. Natl. Acad. Sci. USA 86. supra).
  • mice can be immunized intraperitoneally (I.P.) with a sufficient number of viable cells of the host cell, which is to be transfected by the suppressor gene vector. This can .then be followed immediately by an I.P. injection of, for example, cyclophosphamide in H2O. The cyclophosphamide treatment is repeated one and two days following the primary injection. About two weeks following immunization, mice are then injected with a sufficient amount of the transformed host cells and then allowed to rest for another two weeks, at which time the entire procedure is repeated. Four days following the second injection of the transformed cells, the animals are sacrificed and their spleens obtained for the first fusion.
  • I.P. intraperitoneally
  • Hybridomas are produced by fusing cells by typical techniques, such as from immunized mice with SP2/0 myeloma cells by a polyethylene glycol (PEG) method.
  • Cells are asceptically removed from immunized mice and a single cell suspension of the spleen cells obtained by perfusing the spleen with serum-free media (e.g. DME) .
  • Spleen cells and myeloma cells are mixed together at a ratio, for example, of 5 to 1, spleen cells to myeloma cells.
  • the cells are then centrifuged and the supernatant removed by aspiration.
  • the cells are then grown in medium by standard techniques.
  • Hybridomas which grow after the fusion procedure, are then screened for secretion of anti-human suppressor gene product antibodies by an ELISA assay on a cell lysate. Hybridomas that produce positive results, are expanded and cloned by limiting dilution to assure that the cells and resulting antibodies are indeed monoclonal. Hybridoma colonies, that test positive for the presence of antibody to the human suppressor gene product are diluted in media to a concentration of, for example, 5 hybridoma cells per milliliter. Once colonies grow, the supernatants are again tested for the presence of antibody to the human suppressor gene product. If the results are positive when tested by an ELISA assay, the colonies are cloned again by limiting dilution.
  • the resultant antibodies can then be used as probes in the assay method discussed above.
  • - Antibody probes are particularly sensitive to alterations or deletions in the suppressor gene, such as a PTPase gene, preferably PTPIB, which affect the resultant protein products.
  • the antibody probes provide a simple and efficient means of determining whether a deletion or alteration of the gene has affected a functional unit.
  • By using antibody probes it is possible to determine both the level of expressed protein and whether there has been a change in expression.
  • One can compare results against base line levels obtained for the material being sampled, e.g., level of suppressor gene product in blood, or by comparing a test cell (preselected cell) from an individual with another cell from that individual, which is not believed to show malignancy.
  • test cell samples from the same individual at various times to provide levels of comparison. This can also be done for the nucleotide probes.
  • an antibody or cocktail of probes e.g. antibody probes
  • the probes e.g. antibodies
  • the probes can be labelled directly with a reporter or indirectly with a member of a specific binding pair using conventional techniques.
  • Specific binding pairs can be of the immune or non-immune type.
  • Immune specific binding pairs are exemplified by antigen-antibody systems of hapten/anti-hapten systems. These include fluorescein/anti-fluorescein, dinitrophenyl/anti-dinitrophenyl, biotin/anti-biotin, peptide/anti-peptide and the like.
  • the antibody member of the specific binding pair can be produced by customary methods familiar to those skilled in the art. Such methods involve immunizing an animal with the antigen member of the specific binding pair. If the antigen member of the specific binding pair is not immunogenic, e.g., a hapten, it can be covalently coupled to a carrier protein to render it immunogenic.
  • Non-immune binding pairs include systems wherein the two components share a natural affinity for each other but are not antibodies.
  • Exemplary non-immune pairs are biotin-streptavidin, intrinsic factor-vitamin Bin, folic acid-folate binding protein and the like.
  • Biotin can be covalently coupled to antibodies by utilizing commercially available active derivatives. Some of these are biotin-N-hydroxy-succinimide which binds to amine groups on proteins; bitoin hydrazide which binds to carbohydrate moieties, aldehydes and carboxyl groups via a carbodiimide coupling; and biotin maleimide and iodoacetyl biotin which bind to sulfhydryl groups.
  • Fluorescein can be coupled to protein amine groups using fluorescein isothiocyanate. Dinitrophenyl groups can be coupled to protein amine groups using 2,4-dinitrobenzene sulfate or 2,4-dinitrofluorobenzene. Other standard methods of conjugation can be employed to couple monoclonal antibodies to a member of a specific binding pair including dialdehyde, carbodiimide coupling, homofunctional crosslinking, and heterobifunctional crosslinking. Carbodiimide coupling is an effective method of coupling carboxyl groups on one substance to amine groups on another. Carbodiimide coupling is facilitated by using the commercially available reagent l-ethyl-3-(dimethyl-aminopropyl)-carbodiimide (EDAC) .
  • EDAC commercially available reagent l-ethyl-3-(dimethyl-aminopropyl)-carbodiimide
  • Homobifunctional crosslinkers including the bifunctional imidoesters and bifunctional N-hydroxy-succinimide esters, are commercially available and are employed for coupling amine groups on one substance to amine groups on another.
  • Heterobifunctional crosslinkers are reagents which possess different functional groups.
  • the most common commercially available heterobifunctional crosslinkers have an amine reactive N-hydroxysuccinimide ester as one functional group, and a sulfhydryl reactive group as the second functional group.
  • the most common sulfhydryl reactive groups are maleimides, pyridyl disulfides and active halogens.
  • One of the functional groups can be a photoactive aryl nitrene, which upon irradiation reacts with a variety of groups.
  • the detectably-labelled probe e.g., antibody, detectably-labelled antibodies, or detectably-labelled member of the specific binding pair is coupled to a reporter which can be a radioactive isotope, enzyme, fluorogenic, chemiluminescent or electrochemical materials.
  • a reporter can be a radioactive isotope, enzyme, fluorogenic, chemiluminescent or electrochemical materials.
  • 125 3 isotopes are I and H.
  • Standard radioactive isotopic labeling procedures include the chloramine T, lactoperoxidase and
  • Enzymes suitable for use in this invention include, but are not limited to, horseradish peroxidase, alkaline phosphatase, ?-galactosidase, glucose oxidase, luciferase, ⁇ -lactamase, urease and lysozyme. Enzyme labeling is facilitated by using dialdehyde, carbodiimide coupling, homobifunctional crosslinkers and heterobifunctional crosslinkers as described above for coupling an antibody with a member of a specific binding pair.
  • the labeling method chosen depends on the functional groups available on the enzyme and the material to be labeled, and the tolerance of both to the conjugation conditions.
  • the labeling method used in the present invention can be one of, but not limited to, any conventional methods currently employed including those described by Engvall and Pearlmann, Immunochemistrv 1:871 (1971), Avrameas and Ternynck ' , Immunochemistry 1:1175 (1975), Ishikawa et al., J. Immunoassav 4 (3):209-327 (1983) and Jablonski, Anal. Biochem. 148:199 (1985), which are incorporated by reference.
  • Labeling can be accomplished by indirect methods such as using spacers or other members of specific binding pairs.
  • An example of this is the detection of a biotinylated antibody with unlabelled streptavidin and biotinylated enzyme, with streptavidin and biotinylated enzyme being added either sequentially or simultaneously.
  • the antibody used to detect can be detectably-labelled directly with a reporter or indirectly with a first member of a specific binding pair.
  • detection is effected by reacting the antibody-first member of a specific binding complex with the second member of the binding pair which is labelled or unlabelled as mentioned above.
  • the unlabelled detector antibody can be detected by reacting the unlabelled antibody with a labelled antibody specific for the unlabelled antibody.
  • a labelled antibody specific for the unlabelled antibody can be labelled directly or indirectly using any of the approaches discussed above.
  • the anti-antibody can be coupled to biotin which is detected by reacting with the streptavidin-horseradish peroxidase system discussed above.
  • biotin One preferred embodiment utilizes biotin.
  • the biotinylated antibody Is in turn reacted with streptavidin-horseradish peroxidase complex.
  • Orthophenylenediamine, 4-chloro-naphthol, or tetramethylbenzldine (TMB) can be used to effect chromogenic detection.
  • the preferred immunoassay format for practicing this invention is a forward sandwich assay in which the capture reagent has been immobilized, using conventional techniques, on the surface of the support.
  • Suitable supports used in assays include synthetic polymer supports, such as polypropylene, polystyrene, substituted polystyrene, e.g., aminated or carboxylated polystyrene; polyacrylamides; polyamides; polyvinylchloride, etc.; glass beads; agarose; nitrocellulose, etc.
  • an individual suffering from a cancerous or precancerous condition preferably the ones being diagnosed by the probes by supplying a therapeutic amount of the suppressor gene product.
  • This can be accomplished by a number of methods known in the art. [Williams, D.A., et al, Nature 110:476-480 (1984); Cepko, C.L., et al, Cell 37____:1053-1062 (1984) which are incorporated by reference].
  • gene transfer techniques to prepare a vector containing a nucleotide sequence corresponding to the suppressor gene or functional fragment thereof, preferably a PTPase gene, more preferably PTPIB and use such a vector to transform the malignant cells.
  • the vector is preferably a retroviral vector such as described by Brown and Scott, in DNA Cloning, vol III, A Practical Approach, ch. 9, “Retroviral Vectors", CRL Press (1987), which is incorporated herein by reference.
  • the suppressor gene used is the PTPIB gene or a functional fragment thereof.
  • a PTPase transformed cell line to produce large amounts of the PTPase gene product, e.g., PTPIB gene product, which is then isolated and purified so that it is substantially free of pyrogens and endotoxins.
  • the gene product is preferably purified so that is is at least about 90% pure, more preferably at least about 95% pure, still more preferably at least about 98% pure.
  • the purified protein is then packaged by standard pharamaceutical procedures so that it can be delivered to the malignant cells, such as by injection, carrier-linked preparations, etc.
  • a therapeutically effective amount of the purified protein is used. The therapeutically effective amount can readily be determined empirically based upon the present disclosure.
  • a PTPase IB overexpressing cell line which is subsequently infected by a virus expressing the a neu oncogene, e.g., MLV TM neu-neo virus, has greater resistance to neoplastic transformation than an equivalent cell not having PTPase overexpression.
  • the foci are smaller and the cells appear less morphologically transformed.
  • Figure 6 which illustrates the reduced suseptibility of PTPase IB expressing cells to transformation by the human neu oncogene.
  • the foci induced in NIH3T3 cells (Figure 6B) are larger and denser than the foci induced in 3T3 3-18 cells (6E) and the NIH3T3 cells show a characteristic transformed morphology (6C) , while 3T3 3-18 cells do not (6F) .
  • Figure 6A shows a monolayer of mock infected NIH3T3 cells.
  • Figure 6B shows a monolayer of MLV TM neu-neo virus Infected NIH3T3 cells.
  • Figure 6C shows a G418 resistant colony of MLV TM neu-neo virus infected 3T3 cells.
  • Figure 6D-F are the same as 6A-C but use 3T3 3-18 cells instead of NIH3T3.
  • the antisense oligonucleotide designed from oligopeptide 1 was chosen for further uses as a sequencing primer to identify the 5' end of any open reading f ame in putative cDNA clones.
  • DNA fragments that hybridized to the degenerate oligonucleotides were introduced into the sequencing vector pGEM3Z (Promega) for DNA sequence determination by the chain-terminating method [Sanger, F. , et al, Proc. Natl. Acad. Sci. USA 74:5463-5467 (1977)] using the Sequenase reaction (United States Biochemical) .
  • RNA transcripts were translated in vitro using a rabbit reticulocyte lysate (Promega).
  • the [ JJ S]methionine labelled translation reaction products were displayed by SDS/PAGE [Laemmli, U.K., Nature 227:680-685 (1970)] and visualized by autoradiograph [Ausubel, F.M. , et al, supra] .
  • a human placental cDNA library was screened with degenerate oligonucleotides deduced from the partial amino acid sequence of the PTPase IB as described above.
  • One recombinant bacteriophage was shown by Southern analysis to contain a DNA fragment of 3.2 kb that hybridized to both of the degenerate oligonucleotides. The fragment was subcloned into pGEM3Z by standard techniques, described above and designated pAbT9611.
  • FIG. 7 is a schematic of pAbT 9611, which is a plasmid containing a cDNA clone encoding the entire protein coding region of PTPIB and a bacterial replicon (pGEM3Z, Promega) which codes for an ampicillin resistance gene for growth and selection in E_ coli.
  • a sample of this plasmid has been deposited with the American Type Culture Collection (ATCC) under the Budapest Treaty and given ATCC Accession No. 40768. The DNA sequence was determined.
  • the cDNA sequence of the PTPIB gene, the deduced translation product, and sequences corresponding to the degenerate oligonucleotides used in the cDNA cloning are shown in Figure 1.
  • the observed 1305 nucleotide open reading frame predicts a protein of 435 amino acids of molecular weight 49,966.
  • the amino terminal 321 amino acids encoded by the cDNA are identical to the empirically determined amino acid sequence of the 37000 dalton PTPase IB of Charbonneau, H. , et al., Proc. Natl. Acad. Sci USA 86. supra.
  • the cDNA clone, pAbT9611 was used as a template for in vitro transcription and translation.
  • the major translation product was a protein with an apparent molecular weight of 50,000 as measured by SDS/PAGE.
  • Lane A Radiolabelled molecular weight markers (Amersham) ; Lane B: Translation products from transcription of the cDNA digested with BamHI which cleaves the plasmid at a site 3' to the cDNA insert; Lane C: Translation products from transcription of the cDNA digested with Hindlll which cleaves within the cDNA coding sequence; Lane D: No added
  • the analysis of the cDNA indicates that the primary translation product of the PTPIB gene is a 435 amino acid protein with a predicted molecular weight of 49,966, which is in contrast to the 321 amino acid PTPase IB protein previously purified and sequenced [Charbonneau, H. , et al. Bioch. 86:5252-5256 (1989)].
  • PTPase IB antibody fraction provided by N. Tonks, University of Washington
  • PTPase IB expressed from cDNA clones in NIH 3T3 cells appears to be a 50,000 dalton protein.
  • the 435 amino acid PTPase IB is the functional species, and that the carboxy-terminus may perform a necessary role, perhaps in regulating enzymatic activity, controlling access to substrates, or possibly targeting the protein to the cytoplasmic surface of the membrane.
  • the overall hydrophobic character and conserved amino acids at the carboxy-terminus of the PTPase IB and the T cell PTPase suggests that this region of the protein is important.
  • the fact that 50% of the PTPase IB activity is obtained from the particulate fraction [Tonks, N.K. , et al, J. Biol. Chem. 263. 6722-6730 (1988)] suggests that PTPase IB may be membrane associated.
  • a human genomic library prepared in the lambda vector EMBL-3 Sp6/T7 (Clontech) was screened for recombinant bacteriophage containing sequences that hybridized to the PTPase IB cDNA clone. Approximately 2x10 recombinant bacteriophage were screened as described by Ausubel, F.M. , et al, supra with the probe radiolabelled using [o(- 32 ⁇ ]CT ⁇ in a random hexanucleotide priming reaction [Feinberg, A.P. et al, Anal.
  • Genomic clones were subjected to
  • a genomic clone was isolated by screening a human genomic library with PTPIB cDNA, and designated LAbT10-l. See Figure
  • LAbT10-l is a lambdaphage clone containing 13 kilobases of the human genome that encode a portion of the PTPIB gene.
  • the insert was cloned as a Sau 3A'fragment into the BamHI site of the lambda cloning vector EMBL-3 Sp6/T7.
  • the human DNA replaces the stuffer fragment and the restriction sites for Eco Rl, BamHI and Xba 1 were removed.
  • a sample has been deposited with the ATCC under the Budapest Treaty and given ATCC Accession No. 40767. Two genomic fragments of approximately 13 kb each were isolated. Restriction analysis indicates that the two clones exhibit nearly 90% overlap.
  • a single Hindlll site is present in the cDNA sequence suggesting that there may be an additional, smaller genomic Hindlll fragment not seen on the Southern blots.
  • the genomic clone contains an EcoRI site in the intron separating exons C and D (Fig. 4).
  • a second genomic EcoRI fragment encompassing exons D and E may not have been observed since these exons contain only 218 nucleotides that are homologous to the open reading frame portion of the cDNA.
  • the recombinant lambda phage genomic clone (LAbT 10-1) was used for gene mapping by fluorescence in situ hybridization.
  • the basic procedure for localizing single copy genes with fluoresence detection has been described by Lawrence, J.B., et al, Cell 12:51-61 (1988); Staunton, D.E. , et al, J. Exp. Med. 169:1087-1099 (1989), which are incorporated by reference, and a detailed analysis of banding procedures described by Lawrence, J.B., et al, Science (1990) which is incorporated by reference.
  • the denatured probe previously labelled by nick- ranslation with biotin d-UTP, was present at a final concentration of 5 /-g/ml in the hybridization solution.
  • An excess of non-specific competitors including total human placental DNA was also included to compete hybridization to repetitive sequence elements. Specific hybridization was detected by staining with fluorescein avidin.
  • Metaphase chromosomes were identified by diaminophenylindole (DAPI) banding, enhanced by prior incorporation of 5-bromodeoxyuridine into chromosomal DNA.
  • DAPI diaminophenylindole
  • Giemsa-trypsin banding prior to hybridization was done to confirm these results.
  • the localization of the gene and interpretation of banding patterns in 10-12 metaphases was performed by two independent observers. The localization was also analyzed by simple measurement of signal position relative to the total length of the chromosome in 20 metaphase figures.
  • the deposited genomic clone was nick translated In the presence of biotinylated dUTP and then used to probe human metaphase chromosomes. Localization to a single chromosomal locus was determined by non-isotopic, flourescence detection of in situ hybridization. Over 90% of metaphase figures showed hybridization which was consistently on a chromosomal pair having the small metacentric morphology of F group chromosomes. Chromosome banding analysis was performed primarily by DAPI-staining of BrdUrd-incorporated chromosomes and was confirmed with Giemsa-trypsin banding.
  • Placement of the signal was also analyzed by measurement along the length of the chromosome; based on the average of 27 determinations, the PTPIB gene would map in the middle of ql3.1. However, because banding analysis is more reliable, we conclude that PTPIB is localized to 20ql3.1-13.2.
  • chromosome 20q deletions spanning the qll-ql3.3 region have been observed in some patients with myeloid disorders [Le Beau, M.M. , et al. Proc. Natl. Acad. Sci. USA 82, 6692-6696 (1985); Davis, M.P., et al, Cancer Genet. Cytogenet 12. 63-71 (1984); Mitelman, F. , Cvtogenet. Cell Genet. 16, 1-516 (1984); Testa, J.R., et al, Blood 52. 868-877 (1978)].
  • PTPase IB was cloned into the bacterial expression vector pET-3B (Studier and Moffatt, 1986) expressed from the T7010 promoter and used to transform pLYS S cells an E. coli strain (BL21DE3) containing a T7 polymerase gene expressed from a lac inducible promoter and constitutive expression of lysozyme.
  • Cells were grown overnight in LB media containing 200 ⁇ g/ml ampicillin and 25 ⁇ g/ml chloramphenicol. The cells were diluted 100 fold in LB, 200 ⁇ g amp and grown to an ODg Q of 1. At this point IPTG was added to a final concentration of 0.4/xM and the cells were incubated for an additional 3 hours.
  • Bacterial expressed PTPase IB resulted in the protein being sequestered as insoluble inclusion bodies.
  • the inclusion bodies were then isolated from the bacteria by using the following protocol.
  • Bacterial cells were collected by centrifugation and subjected to three cycles of freeze-thaw.
  • the thawed cells were resuspended in 1 ml of ST (lOmM Tris pH8, lOOmM NaCl) containing 0.05% NP40, 2.5mM MgCl 2 , lmg/ml DNAse per 10 ml of culture.
  • ST lOmM Tris pH8, lOOmM NaCl
  • the resulting suspension was incubated on ice for 15 minutes and then sonicated for 1 minute.
  • the suspension was then centrifuged at 10,000xg for 10 minutes.
  • the pellet was resuspended in 1 ml of Hermans buffer (lOmM Tris pH 7.2, lOOmM NaCl, ImM EDTA, 1% NP40, 0.5% Nadeoxycholate) vortexed and centrifuged at 10,000xg for 10 minutes.
  • the pellet was resuspended in 1ml of Hermans containing 2mM beta-mercaptoethanol(BME) and centrifuged at 10,000xg for 10 minutes.
  • the resulting pellet was resuspended in 1ml of 25mM Tris pH8, 2M NaCl, 2mM BME and centrifuged in 10,000xg for 10 minutes.
  • the resulting pellet was resuspended in 100/.1 of TE.
  • Figure 9A shows the protein content of the inclusion bodies and is a coomasie blue stained gel of the PTPase IB inclusion body material.
  • the first lane shows the material before solubilization by the addition of of 6M guanidinium-HCL, 5mM Dithiothreitol (DTT) (starting material) and the remaining lanes, after the guanldine was dialyzed out of the sample (post-dialysis) .
  • the slowest migrating band represent intact PTPase IB protein.
  • the other lower bands are a mixture of contaminating bacterial proteins and proteolytic degradation products of PTPase IB. After dialysis to remove the guanidine approximately 50% of the intact PTPase IB remained soluble.
  • mice were then used as an immunogen.
  • Four mice were immunized with 50 g of inclusion body protein and then boosted at day 14 and day 36 with 50xg of the same Inclusion body material, and fused on day 40. These mice generated a response that recognized the inclusion body materials in an ELISA.
  • 96 well plates were coated with 0.1/-g/ml of either PTPase IB inclusion body material or uninduced lysates in 0.05M Carbonate buffer, pH 9.6 overnight at 4°C.
  • the protein solution was aspirated off and 50 ⁇ g of hybridoma supernatant, immunized mouse sera diluted 1 to 1000 in blotto or normal mouse sera diluted 1 to 1000 in blotto was added and incubated at 37°C for 90 minutes.
  • the plates were washed 3 times with PBS containing Tween 20 and then incubated with 50 ⁇ l with HRP-GAM (Pierce Chemical Company) diluted 1 to 5000 in blotto for 1 hour at 37°C.
  • the plates were washed 3 times with PBS containing Tween 20 and developed with TMB (Transgenic Sciences Inc.) for five minutes and stopped with 2.5N sulphuric acid.
  • the OD was read at 450nm.
  • hybridomas resulted in two monoclonal antibodies (MAbs) AE4-2J (ATCC Accession No. HB10666) and CE5-1K (ATCC Accession No. HB10667) .
  • MAbs monoclonal antibodies
  • CE5-1K ATCC Accession No. HB10667
  • a second fusion was performed using an additional mouse boosted as described above with the following exceptions, one additional boost on day 70, the final boost was on day 174 with fusion on day 177.
  • 197 hybridomas were screened by western blot analysis against PTPIB protein expressed from vAbT 477-2-2 (described below). Thirty-nine of the hybridomas screened by western blot reacted with the 49K PTPIB protein. These hybridomas were single cell cloned and rescreened by western blot analysis. Twenty-two of the primary clones were positive and were subjected to a second round of single cell cloning and screening by western blot. This resulted in 19 new hybridoma cell lines.
  • the isotypes of the antibodies were determined using the Amersham
  • AE4-2J IgGl This analysis permits classification of these antibodies into five classes that react with unique epitopes.
  • the class represented by the antibody FG6-lg (ATCC Accession No. HB 10690) reacts with an epitope in the 37kd core domain of PTPaselB that is also present in T cell PTPase.
  • the class represented by the antibody CA6-ld (ATCC Accession No. HB 10691) reacts with a different epitope in the 37kd core domain of PTPaselb and does not cross react with T cell PTPase.
  • the remaining three classes represented by the antibodies DH8-lb (ATCC Accession No.
  • AE4-2J and CE5-1K recognize unique epitopes in the carboxy terminal domain of PTPase IB and do not cross react with T cell PTPase. These three antibodies are distinguished from each other by their cross reactivity to other unidentified proteins from either vaccinia or the BSC-40 cells used for the infections.
  • 3T3, 3-18 and 3-18 neu cells were lysed in hypotonic lysis buffer containing protease inhibitors. Approximately 90 ⁇ g of each of the cell lysate was run on SDS-PAGE gels and blotted onto nitrocellulose. The blots were probed with undiluted supernatant from the hybridoma cell lines. See Figure 10. These antibodies detect a 49kD protein in 3-18 cells, but not in 3T3 cells. MAb AE4-2J also detected a protein of approximately 90kD. This protein was detected in both mouse cells and human cells.
  • Figure 11 indicates that AE4-2J, FG6-lg and DH8-lb recognize a 49kD protein in 3-18 cells but not 3T3 cells. CE5-lk appears to weakly immunoprecipitate this 49kD protein and CA6-ld does not detect this protein at all. The Mab AE4-2J did not appear to immunoprecipitate the 90kD protein seen in the western blot analysis.
  • the entire PTPase IB coding region was isolated as a 1.4 Kb BamHI/Sphl restriction fragment.
  • the Sph I site was made blunt using T4 polymerase.
  • This fragment was subcloned into Bam Hl/Sma I digested pAbT4587 ( Figure 16).
  • pAbT4587 is 4069 base pairs and derived from pAG3 and vaccinia Hind M frag plus the 40K promoter.
  • the resulting plasmid, pAbT 9110 ( Figure 17A) expresses PTPase IB from the vaccinia 40 K promoter.
  • T cell PTPase coding region was isolated as a 2.3 Kb Eco Rl restriction fragment [Cool, et al. , Proc. Nat. Acad. Sci.. USA 86:5257 (1989)]. This fragment was subcloned into Eco Rl digested pAbT4587 ( Figure 16) . The resulting plasmid, pAbT 9112 ( Figure 17B) expresses T cell PTPase from the vaccinia 40 K promoter.
  • Vaccinia virus has been developed as an infectious eukaryotic cloning vector.
  • PTPase IB and T cell PTPase have been expressed in vaccinia by the method of Mackett, M. , et al. , J. Gen. Virol. 67:2067 (1986) except the selection scheme for the recombinant virus was a host range selection, Smith, K. , PCT 89102486.
  • Recombinant vaccinia virus vAbT33 contains the lacZ gene in place of a portion of the
  • IVR vector pAbT9110 was transfected into BSC-40 cells which had been infected with vaccinia virus vAbT33. Viral infection and plasmid transfection were performed essentially as described in Spyropoulos, et al., J. Virol. 62:1046 (1988). Recombinant viruses were selected as white plaques in the presence of Bluogal on RK-13 cells. Plaques were picked and purified, and the final recombinant, designated vAbT477, was amplified on RK-13 cells and purified over a 36% sucrose cushion.
  • the BSC-40 cells infected with either PTPIB (vAbT 477-2-2) or NYCBH, a wild-type vaccinia virus were lysed in hypotonic lysis buffer containing protease inhibitors. Approximately 20 ⁇ g of each lysate was run on SDS-PAGE gels and blotted onto nitrocellulose. The blots were probed with undiluted hybridoma supernatant from either of the two Mabs (See Figure 10).
  • Lysates of BSC-40 cells infected with either PTPIB (477-2-2) or NYCBH were run on polyacrylamide gels and transferred to nitrocellulose.
  • the blots were cut into strips and probed with sera from immunized mice, PTP IB, normal mouse sera (NMS) or hybridoma supernatant from the PTP IB MAbs AE4-2J or CE5-1K. See Figure 12.
  • the sera was diluted 1:1000 in Blotto and the supernatant was used straight.
  • the line in all the strips at 200 kD is an orientation mark used to align the strips. (See Figure 12).
  • the PTP IB hybridoma supernatant detected a ca. 50 kD protein that was specific to cells infected with PTPIB. In all cases, the sera did not react with lysate from cells infected with NYCBH virus and normal mouse sera did not react with the vaccinia expressed PTPase proteins (See Figure 12) .
  • the T cell PTPase cDNA was inserted into the Hindlll M region of vaccinia virus using the same methodology described above for PTPase IB.
  • the IVR vector pAbT9112 was used resulting in the recombinant vaccinia virus vAbT 479.
  • Expression of the T cell PTPase was confirmed by western blot analysis of VABT479 infected BSC-40 cells using antisera raised by immunizing mice with T cell PTPase protein purified from recombinant baculo virus infected insect cells.
  • PTPase Overexpression of a PTPase is capable of inhibiting cellular transformation.
  • a cell line overexpressing PTPase was prepared as indicated below. The PTPase overexpressing cell line was then infected with an oncogenic retrovirus and the infected cells were analysed for parameters of transformation.
  • NIH 3T3 cells were cotransfected with a plasmid that expresses PTPase IB (pAbT9703) from the MLV LTR promoter and a plasmid that expresses the gene encoding hygromycin resistance pTKhygro from the Herpes virus thymidine kinase promoter. Transfected cells were then plated in the presence of lOOug/ml hygromycin and 22 colonies were isolated. The selected colonies were then expanded and RNA was isolated from cells derived from each colony.
  • PTPase IB pAbT9703
  • pTKhygro Herpes virus thymidine kinase promoter
  • RNA dot blot analysis was performed using a fragment containing the PTPase IB coding region from pAbT9611 as the probe and one colony showed high levels of PTPase IB mRNA. This colony was designated 3T3 3-18 and PTPase IB expression was confirmed by Western analysis using an antibody fraction supplied by Dr. Edmund Fischer of the University of Washington.
  • MLV TM-neu neo virus expresses an allele of the human neu oncogene which includes a point mutation in the transmembrane domain that enhances transforming activity.
  • the transforming activity of neu is thought to be due to its tyrosine kinase activity and oncogenic activation by the transmembrane point mutation results in an increase in tyrosine kinase activity.
  • MLV TM-neu neo expresses the gene for G418 resistance. Infectious, replication defective viral stocks are derived from Psi 2 cells transfected with the plasmid pAbT9003.
  • PTPase activity was assayed by measuring the release of orthophosphate from tyrosine-phosphorylated RCM lysozyme, Tonks, N.K. , et al, J. of Biol. Chem.. 263:14. 6722-6730 (1988).
  • Control cells or retrovirally infected cells expressing neu TM were harvested in a hypotonic buffer without detergents and lysed by dounce homogenization. After centrifugation, the supernatant fraction, referred to as the "soluble" fraction, was reserved and the insoluble sediment was resuspended in buffer containing 1% Triton.
  • PTPase activity that is two to three times over endogenous activity. The majority of the excess PTPase activity is found in the Triton soluble fraction of cell lysates which is similar to that observed with endogenous activity.
  • Both PTPase-expressing 3-18 cells and the parental NIH3T3 cells were plated at a density of 1.5X10 cells per 60 mm dish. The following day the cells were either mock infected or infected with MLV TM-neu neo virus. Eighteen hours later the cells were split into normal growth media and growth media containing 800ug/ml G418. After 10 days the plates were analysed for the presence of foci or G418 resistant colonies, respectively. Both infected NIH 3T3 and 3T3 3-18 cells give rise to G418 colonies, but only the NIH 3T3 colonies were morphologically altered (Figure 6B, E) . In the focus assay both cell lines gave rise to foci but the 3T3 3-18 foci were smaller and the cells in the foci appeared to be less morphologically transformed ( Figure 6C, F) .
  • Neu protein levels were determined by capture ELISA using two monoclonal antibodies (designated NB3 and TAl) directed against the neu gene product, for two individual G418 colonies from both NIH 3T3 and 3T3 3-18. Although the 3T3 3-18 colonies are not morphologically transformed, they contain active neu protein. This indicates that the resistance to transformation is due to PTPase expression and not decreased neu expression.
  • the NIH 3T3 cell lines, 3T3 neu TM cell lines (3T3 cell lines transformed by the MLV TM-neu expressing virus), 3-18 hygro uninfected cell lines, 3-18 hygro/neu TM cell lines, (3-18 hygromycin sensitive cell lines transformed by the neu expressing TM virus) and 3-18 TM neu cell lines were suspended in 0.35% agar and grown for 28 days. Macroscopically observable colonies were counted on an illuminated colony counter. The 3-18 hygro uninfected and 3-18 hygro/neu TM cell lines were passaged in media containing hygromycin prior to plating in soft agar.
  • the 3-18 neu TM cells were passaged In the absence of hygromycin.
  • the results of this soft agar assay are set forth below.
  • Nude mice were subcutaneously injected with either control uninfected cells or cells previously infected with the retrovirus expressing neu TM. Cells infected with MLV TM neuneo were expanded under selection, G418 (800 ⁇ g/ml) for 3T3 neu.
  • mice were injected, as described above, with 3-18 cells infected with the neu TM retrovirus, and again, developed large tumors.
  • the mice injected with infected 3-24 cells only exhibited small tumors. See, Table 2 above. Tumors were excised from the mice, which had received the 3-18 neu TM cells, and examined by Western blot analysis, for PTPase IB expression. (See, Table 3, experiment 2 below) . Although initially 3-18 cells show higher levels of PTPase IB then 3-24 cells, the tumor derived 3-18 cells showed no observable expression of PTPase IB except for one small tumor, which showed a slight PTPase expression upon examination of twice as much tumor sample.
  • MAb AE4-2J was used to probe cell lysates from neu TM infected cells or tumor derived cells, which had been electrophoresed and transfered to nitrocellulose. See Figure 13.
  • Lanes 1 and 2 are uninfected control cell lysates (3T3 and 3-18 respectively) .
  • Lanes 3-5 are lysates of infected cells before injections into mice.
  • Lanes 6-11 are lysates of tumor derived cells from each set of injections.
  • the arrows on the right-hand side of the page indicate MW weight, whereas the arrow on the left-hand side of the page indicates PTPase IB.
  • the 3-18 neu TM tumor samples showed no detectable PTPase IB expression and this result was independent of the application of selective pressure to 3-18 neu TM cells. See Figure 13 lanes T3 to T6. Western analysis also showed high levels of neu TM expression in all tumor samples as did a neu ELISA. (See, Table 3, experiment 3).
  • NIH 3T3 cells were transfected with the MT promoter-PTPlB plasmid 5AbT 9710 ( Figure 18) .
  • This plasmid was constructed by isolating an SacII/Cla I fragment that contains the entire PTPase IB coding region from the plasmid pAbT9703. The SacII site was blunted using the Klenow fragment of E. coli DNA polymerase I. After the addition of Cla I linkers the PTPase IB fragment was subcloned into the unique Cla I site of the pNUTC vector.
  • the resulting plasmid, pAbT9710 contained a PTPase IB cDNA expressed from the heavy metal inducible Mtl-1 promoter and a DHFR gene for selection in methotrexate. 10 ⁇ g of plasmid DNA was used to transfect 3T3 cells using standard calcium phosphate transfections. Individual colonies were isolated by selection in lOOnM methotrexate (MTX) and tested for inducible PTPase IB expression. After overnight growth in various concentrations of either zinc or copper, the clones were examined by RNA dot blot analysis for levels of PTPIB expression. Specific and sharp induction of PTPIB occurs at 80 ⁇ M zinc.
  • MTX methotrexate
  • the transfected cell lines were examined by Western analysis for zinc inducible PTPase IB expression. Clones were obtained which exhibit no basal (uninduced) level and no induced expression, or higher basal expression than desired, as well as one clone which had the desired low basal expression and induction of PTPase IB expression to at least the same level as constitutively expressed PTPase IB in 3-18 cells. This clone was called 43-5. Cell lysates of these cells were prepared by sonication in a Triton X-100 buffer and 150 ⁇ g of protein loaded onto a 12% polyacrylaraide gel. After electrophoresis, proteins were transferred to nitrocellulose and probed with an anti-T cell PTP rabbit antisera. Lysates were prepared from NIH 3T3 and 3-18 cells as negative and positive controls, respectively. See, Figure 14. The arrow in Figure 14 indicates PTPase IB.
  • NIH 3T3 cells or PTPase IB expressing cell lines (3-18 and 3-24) were infected with neu TM as described above, lysed in the presence of the PTPase inhibitor, sodium orthovanadate, and immunoprecipitated with the h-neu specific antibody, PB3,
  • Figure 15 shows that the neu TM-infected 3T3 cells (lanes 2 and 3) had high levels of autophosphorylation on the neu TM molecule pl ⁇ S 2 ⁇ , while neu-infected 3-18 or 3-24 cell lines, lanes 5-6 and 7-8, respectively, exhibit 1/2 to 2/3 less pl ⁇ S 2 ⁇ autophosphorylation.
  • the arrow in the figure indicates pl85.

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Abstract

Segments nucléotides et sondes de nucléotides de phosphotyrosyl phosphatase protéique (PTPase). De préférence, le gène PTPase est PTP1B. L'invention concerne également leur utilisation dans les méthodes de diagnostique.
PCT/US1991/001432 1990-03-02 1991-03-01 METHODES DE DIAGNOSTIQUE UTILISANT DES GENES PTPase ET LEURS UTILISATIONS WO1991013173A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994003611A2 (fr) * 1992-08-05 1994-02-17 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Sous famille ptp-d de tyrosine phosphatases de proteines
WO1995006735A2 (fr) * 1993-09-01 1995-03-09 The Ludwig Institute For Cancer Research Sequences nucleotidiques servant a coder de nouvelles tyrosine-phosphatases proteiques
WO2000053801A1 (fr) * 1999-03-08 2000-09-14 Merck Frosst Canada & Co. Mesure de cellules entieres pour des proteine tyrosine phosphatases
EP2531616A1 (fr) * 2010-02-03 2012-12-12 Monash University Dosage de diagnostic et de pronostic pour le cancer du sein
WO2016042137A1 (fr) * 2014-09-19 2016-03-24 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédé pour diagnostiquer la leucémie myélomonocytique chronique myéloproliférative ou le néoplasme myélodysplasique myéloprolifératif non classifié

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
Proceedings of the National Academy of Sciences (USA), Vol. 86, issued July 1989, COOL et al., "cDNA Isolated from a Human T-Cell Library Encodes a Member of the Protein tyrosine-phosphatase Family", pages 5257-5261, see pages 5257, column 2, paragraph 2. *
Proceedings of the National Academy of Sciences (USA), Vol. 86, issued July 1989. CHARBONNEAU et al., "Human Pacenta Protein-tyrosine-phosphatase: Amino Acid Sequence and Relationship to a Family of Receptor-like Proteins", pages 5252-5256, see entire document. *
See also references of EP0471825A4 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5831009A (en) * 1992-08-05 1998-11-03 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Protein phosphotyrosine phosphatases PTP-D1
WO1994003611A3 (fr) * 1992-08-05 1994-03-31 Max Planck Gesellschaft Sous famille ptp-d de tyrosine phosphatases de proteines
US5955592A (en) * 1992-08-05 1999-09-21 Max Planck Gesellschaft Zur Forderung Der Wissenschaften E.V. Nucleic acid encoding novel protein phosphotyrosine phosphatase PTP-D1
WO1994003611A2 (fr) * 1992-08-05 1994-02-17 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Sous famille ptp-d de tyrosine phosphatases de proteines
WO1995006735A3 (fr) * 1993-09-01 1995-06-22 Ludwig Inst Cancer Res Sequences nucleotidiques servant a coder de nouvelles tyrosine-phosphatases proteiques
US5821075A (en) * 1993-09-01 1998-10-13 The Ludwig Institute For Cancer Research Nucleotide sequences for novel protein tyrosine phosphatases
AU683299B2 (en) * 1993-09-01 1997-11-06 Ludwig Institute For Cancer Research Nucleotide sequences for novel protein tyrosine phosphatases
WO1995006735A2 (fr) * 1993-09-01 1995-03-09 The Ludwig Institute For Cancer Research Sequences nucleotidiques servant a coder de nouvelles tyrosine-phosphatases proteiques
WO2000053801A1 (fr) * 1999-03-08 2000-09-14 Merck Frosst Canada & Co. Mesure de cellules entieres pour des proteine tyrosine phosphatases
US6329137B1 (en) 1999-03-08 2001-12-11 Merck Frosst Canada & Co. Intact cell assay for protein tyrosine phosphatases using recombinant baculoviruses
EP2531616A1 (fr) * 2010-02-03 2012-12-12 Monash University Dosage de diagnostic et de pronostic pour le cancer du sein
EP2531616A4 (fr) * 2010-02-03 2013-07-10 Univ Monash Dosage de diagnostic et de pronostic pour le cancer du sein
WO2016042137A1 (fr) * 2014-09-19 2016-03-24 INSERM (Institut National de la Santé et de la Recherche Médicale) Procédé pour diagnostiquer la leucémie myélomonocytique chronique myéloproliférative ou le néoplasme myélodysplasique myéloprolifératif non classifié

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