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WO1994021791A1 - Agents de prevention et de traitement du cancer du sein - Google Patents

Agents de prevention et de traitement du cancer du sein Download PDF

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Publication number
WO1994021791A1
WO1994021791A1 PCT/EP1994/000651 EP9400651W WO9421791A1 WO 1994021791 A1 WO1994021791 A1 WO 1994021791A1 EP 9400651 W EP9400651 W EP 9400651W WO 9421791 A1 WO9421791 A1 WO 9421791A1
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protein
seq
gene
sequence
nucleic acid
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PCT/EP1994/000651
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English (en)
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Johanna Eugenie Bergmann
Rick Enrique Preddie
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Johanna Eugenie Bergmann
Rick Enrique Preddie
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Publication of WO1994021791A1 publication Critical patent/WO1994021791A1/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to therapeutic agents for the prevention and treatment of breast cancer, especially in humans. More specifically, the invention relates to two genes, BC534 and BC538, that are implicated in causing breast cancer, to antagonists of these genes and their gene products. The invention additionally relates to nucleic acid molecules that influence the expression of these genes. The invention also relates to therapeutic methods that employ all such agents.
  • breast cancer As an inherited trait breast cancer is one of the most common genetic diseases in the industrial world; in fact, one out of 100 women alive today will develop breast cancer due to inheritance unless a cure for the disease is found. About 40% of cases are diagnosed before the patient has attained the age of 30. Only about 5% of all breast cancer is, however, inherited. The remaining 95% of breast cancers result from the acquisition of mutations. Breast cancer is presently treated using surgery, endocrine therapy and chemotherapy (Salmon, S.E., Semin. Oncol.. 12:50-52 (1990); Hortobagyi, G.N., Breast Cancer Res. Treat 2_1:3-13 (1992) .
  • Endocrine therapy results in complete or partial remissions in only 30% of patients (Jiang, S.-Y., et al.. J. Natl. Cane. Inst. 84:580-591 (1992); Muss, H.B., Breast Cancer Res. Treat 21:15-26 (1992) ) .
  • Chemotherapy despite the development of new antineoplastic agents, has had only limited success in treating breast cancer (Hortobagyi, G.N., Breast Cancer Res. Treat 2_1:3-13 (1992)) .
  • surgery is the only presently proven treatment for breast cancer; its use has long been controversial (Albert, S. et al.. Cancer 41:2399-2408 (1978)) . Surgery is often combined with endocrine therapy or chemotherapy regimes.
  • Biological agents such as interferon and interleukin, have been found to be capable of producing definite anti-tumor responses. Unfortunately, such advances have not yet led to improved regimens for managing breast cancer (Hortobagyi, G.N., Breast Cancer Res. Treat 21:3-13 (1992)) . Monoclonal antibodies have also been employed, however, the available antibodies have proven to lack sufficient sensitivity or specificity to selectively target tumor cells (Hortobagyi, G.N., Breast Cancer Res. Treat 21:3-13 (1992) ) .
  • One mechanism through which cancer may arise is through a cell's exposure to a carcinogenic agent, either chemical or radiation. Such exposure may damage the DNA sequence of critical genes present in the genome of a cell of an animal. If this damage leads to either an impairment in the expression of the gene, or in the production of a mutant gene product, the cell may then proceed to proliferate, and ultimately result in the formation of a tumor.
  • a carcinogenic agent either chemical or radiation.
  • Such exposure may damage the DNA sequence of critical genes present in the genome of a cell of an animal. If this damage leads to either an impairment in the expression of the gene, or in the production of a mutant gene product, the cell may then proceed to proliferate, and ultimately result in the formation of a tumor.
  • Oncogenes are genes which are naturally in an “inactivated” state, but which, through the effect of the DNA damage are converted to an "activated” state capable of inducing tumorigenesis (i.e. tumor formation). Oncogenes have been identified in 15-20% of human tumors. The products of oncogenes (“oncoproteins”) can be divided into two broad classes according to their location in the cell.
  • Oncogene products which act in the cytoplasm of cells have readily identifiable biochemical or biological activities (Green, M.R., Cell 56:1-3 (1989)). Those that act in the nucleus of a cell have been more difficult to characterize.
  • Some nuclear oncoproteins such as E1A and myc have transcriptional regulatory activity, and are believed to mediate their activities by the transcriptional activation of cellular genes (Kingston, R.E., Cell 41:3-5 (1985)) .
  • Other nuclear oncoproteins appear to have a complex array of activities (such as DNA binding activity, ability to initiate viral DNA synthesis, ATPase activity, helicase activity, and transcriptional regulatory activity) (Green, M.R., Cell 56:1-3 (1989)).
  • anti-oncogenes or "tumor-suppressing genes.” In their natural state these genes act to suppress cell proliferation. Damage to such genes leads to a loss of this suppression, and thereby results in tumorigenesis. Thus, the deregulation of cell growth may be mediated by either the activation of oncogenes or the inactivation of tumor-suppressing genes (Weinberg, R.A. , Scientific Amer.. Sept. 1988, pp 44-51).
  • Oncogenes and tumor-suppressing genes have a basic distinguishing feature.
  • the oncogenes identified thus far have arisen only in somatic cells, and thus have been incapable of transmitting their effects to the germ line of the host animal.
  • mutations in tumor-suppressing genes can be identified in germ line cells, and are thus transmissible to an animal's progeny.
  • the "p53" gene encodes a nuclear protein that forms a stable complex with both the SV40 large T antigen and the adenovirus E1B 55 kd protein. The p53 gene product may be inactivated by binding to these proteins.
  • the p53 gene was thought to be an oncogene since it is capable of immortalizing primary rodent cells and can cooperate with the ras oncogene to cause transformation. Subsequent research, however, has revealed that the p53 genes used in those early experiments was a mutant allele of the normal p53 gene (Green, M.R., Cell 5j6:l-3 (1989)), and that the p53 gene is a tumor-suppressing gene rather than an oncogene (Green, M.R., Cell 5_6.:l-3 (1989); Mowat et al. , Nature 114:633-636 (1985) ) .
  • the p53 gene has been implicated as having a role in colorectal carcinoma (Baker, S.J. et al.. Science 244:217-221 (1989)) .
  • the region deleted was subsequently found to encompass the p53 gene locus (Baker, S.J. et al.. Science 244:217-221 (1989)) .
  • the deletion of the region was found to mark a transition from a (benign) adenocarcinoma stage to a (malignant) carcinomatous stage (Vogelstein, B. et al.. New En ⁇ l. J. Med.
  • Figure l presents the cDNA and encoded amino acid sequences of BC534, BC538, BC538.1 and BC53/reg.
  • Figure 1A shows the nucleotide sequence of BC534 cDNA. The sequence is 100% homologous to nucleotides 494-559 inclusive on the antisense strand of p53 mRNA (nucleotides 12,204-12269 inclusive on the antisense strand of p53 gene) .
  • Figure IB shows the 2.35 kd, 21 amino acid nuclear protein encoded by the BC534 sequence.
  • Figure 1C shows the nucleotide sequence of BC538 cDNA. The sequence is 100% homologous to nucleotides 82-216 inclusive on the antisense strand of p53 mRNA .
  • BC538.1 includes the first 30 nucleotides of BC538 and the first 222 nucleotides from the intron which seperates BC538 exon #1 and Exon#2.
  • Figure IF shows the 9.27kD protein encoded by BC538.1.
  • Figure 1G shows the transcription regulatory region ("BC53/reg") of the BC53 family of genes.
  • the regulatory region is 100 % homologous to the region of the antisense strand of p53 gene consisting of nucleotides 12,411, 12,518 inclusive. It contains a "CCAAT box” (nucleotide 12,570-12,581), two “TATA boxes” ("1" nucleotide 12,481-12,499 and "2" 12,411-12,570) and a cap site (nucleotide 12,463-12,470).
  • the CCAAT box, one TATA box "1" and the cap site are correlated.
  • Figure 2A/B shows the organisation of the BC53 family of genes including location of "AATAA" transcription termination signals.
  • the invention concerns agents and methods for the diagnosis and therapy of breast cancer and related conditions.
  • agents include antisense molecules capable of influencing the transcription of either of the genes, BC534, BC538 and BC538.1.
  • the invention also includes antagonists of the products of these genes.
  • the invention provides a nucleic acid molecule, substantially free of natural contaminants, that encodes a protein selected from the group consisting of BC534, BC538 and BC538.1.
  • the invention also provides a protein, substantially free of natural contaminants, selected from the group consisting of a BC534 gene product, a BC538 gene product and a BC538.1 gene product.
  • the invention also provides a reagent capable of diagnosing the presence of a molecule selected from the group consisting of a BC534 gene sequence, a BC534 mRNA transcript, a BC534 gene product, a BC538 gene sequence, a BC538 mRNA transcript, a BC538 gene product, a BC538 mRNA transcript and aBC538.1 gene product.
  • the reagent is a nucleic acid molecule (particularly a ribozyme produced from nucleic acid molecules having a sequence of BC534, BC538, BC538.1 or BC53/reg or a nucleic acid molecule obtainable by mutating a nucleic acid molecule having a sequence of BC534, BC538, BC538.1 or BC53/reg or which comprises a nucleic acid sequence that is complementary to the nucleotide sequence of BC534, BC538,BC538.1 or BC53/reg) or a protein (such as an antibody, or a fragment of an antibody, especially one which is capable of binding to a BC534, BC538 or BC538.1 gene product.
  • a nucleic acid molecule particularly a ribozyme produced from nucleic acid molecules having a sequence of BC534, BC538, BC538.1 or BC53/reg or a nucleic acid molecule obtainable by mutating a nucleic acid molecule having a sequence of BC534, BC538, BC538.1 or
  • the invention also provides a method of treating breast cancer which comprises providing to an individual, in need of such treatment, an effective amount of an inhibitor of BC534, BC538, BC538.1 or BC53/reg especially wherein the inhibitor is a protein (such as an antibody, or fragment thereof) or a nucleic acid molecule.
  • the phosphoprotein p53 localise to chromosome 17pl3 was the first confirmed inherited human breast cancer antigen, and is the confirmed factor in about 1% of all inherited cancers (Malkin, D. et al.. Science 250:1233- 1238 (1990) ; Frevier, T. et al.. J. Clin. Invest. 9J):1637-1641 (1992)) .
  • One very strong possible is believed to be a 17 ⁇ hydroxy steroid dehydrogenase
  • the present invention relates to the discovery of three genes, BC534, BC538 and BC538.1 which encode proteins with the potential to cause tumors in humans.
  • the present invention provides the sequence of these molecules, as well as that of the proteins encoded by them. These molecules may be used in the diagnosis, prediction and treatment of breast cancer.
  • the BC534, BC538 and BC538.1 genes are antisense to regions of the human p53 gene, and thus their transcripts cannot be expressed under circumstances under which the p53 gene is normally expressed.
  • BC534, BC538 and BC538.1 genes thus cannot be expressed in healthy humans.
  • the BC534, BC538 and BC538.1 genes can be expressed, however, if mutations or rearrangements occur in the p53 gene which impair or prevent the transcription of that gene. Silent mutations which might occur in the BC534, BC538 or BC538.1 genes will also influence the expression of the antisense protein. Pathogens and toxic substances to which the p53 gene might be exposed can also cause expression of BC534, Bc538 and BC538.1, but in these cases expression of the latter genes will stop once the pathogen/toxic substance is removed from the intracellular environmen .
  • the BC534 cDNA sequence is shown in Figure 1A (SEQ ID NO:l) and the deduced sequence of the protein in Figure IB (SEQ ID NO:2) .
  • the BC534 gene sequence ( Figure 1A; SEQ ID NO:l) is 100% homologous to nucleotides 494-559 inclusive on the antisense strand of p53 mRNA.
  • the 2.35 kd, 21 amino acid biologically active peptide with a nuclear transport signal (nuclear protein) encoded by the antisense cDNA is shown in Figure IB (SEQ ID NO:2) .
  • This protein can affect the intracellular regulation of the following human cancer- related genes/gene products: (1) met-proto oncogene, (2) oncogene related tyrosine kinase receptor Fit-gene which maintains normal conditions in lung, placenta, liver, kidney, heart and brain, (3) c-alb gene proto oncogene tyrosine kinase which is involved in chronic myelogranulous leukemia, (4) human neurofibromitosis type 1 gene, (5) papilloma virus El-1 protein, which is involved in the pathophysiology of genital cancers in humans, (6) estrogen sulfotransferase, which appears to control the level of estrogen receptor by sulfurylating estradiol; it has been demonstrated that mutations in the gene for the androgen receptor (a gene analogous to the estrogen receptor) which prevent proper interaction of androgen with the receptor can cause male breast cancer as well as androgen insufficiency, and (7) human lung surface active protein which is involved in neon
  • the BC538 gene sequence ( Figure 1C; SEQ ID NO:3) is 100% homologous to nucleotides 82-216 inclusive on the antisense strand of p53 mRNA.
  • the 4.98 kd phosphoprotein encoded by the antisense cDNA is shown in Figure ID (SEQ ID NO: ) .
  • the protein has a protein kinase signature (GXGXXG) , sites for cAMP and cGMP dependent phosphorylation, protein kinase c and casein kinase phosphorylation.
  • the protein is a potential activator of (1) human basic conserved protein, (2) wnt-5b protein precursor, (3) int-2 proto oncogene protein precursor, (4) erbb-3 receptor protein tyrosine kinase, (5) zinc finger protein HF10, (6) rb protein, (7) galactosyl transferase kinase, (8) acidic and (9) basic fibroblast growth factor receptor precursor which are involved in epithelial cell tumors, (10) a negative regulator of mitosis which inhibits cells from entering mitosis and prevents cells from leaving mitosis, (11) thyroid peroxidase, and (12) myelo peroxidase. All the above proteins, except myelo peroxidase, have been shown to
  • the BC538.1 gene sequence ( Figure IE; SEQ ID NO: 5) is 100% homologous to nucleotides 11467-11718 (this includes nucleotide 82-112 on the of p53 mRNA) on the antisense strand of p53 DNA.
  • the 9.27 kd phosphoprotein encoded by the antisense cDNA is shown in Figure IF (SEQ ID NO: 6) .
  • the protein has a protein kinase "GXGXXG” signature, and by similarities appears to have leukotriene-like properties . It can affect the regulation of a DNA polymerase, a toxic cysteine protease, thyroid peroxidase, the activating and the endoprotease cleaving enzymes of the ubiquitin pathway, galactosyl transferase protein kinase, granulin/annexin (a disintegrin-like gene, localized at chromosome 17q/21.3, is somatically rearranged in some primary breast cancers) , a member of the annexin family, and the human stem cell protein which is involved in t-cell leukemia and other hemopoetic malignencies.
  • the BC53 gene family 5'- regulatory sequences ( Figure 1G; SEQ IC NO:7) are 100% homologous to nucleotides 12411-14581 inclusive on the antisense strand of the p53 gene.
  • the 5'- regulatory region contains two distinct promoter systems PI and P2.
  • PI is a correlated CCAAT/TATA/CAP promoter system and P2 is a TATA box.
  • the dual promoter system indicates that BC53 family genes must be subject to different transcriptional activating signals as is the case with expression of the same gene in different type tissue.
  • BC534/BC538/BC538.1 genes will not allow expression of these genes in healthy humans,- however, acquired mutations in regions of the p53 gene involved in the antisense repression of these genes will allow BC534/BC538/BC538.1 to be expressed, and thus result in tumor formation.
  • p53 has been described as a "policeman" against tumors, especially virus-induced tumors, in humans, and, at least in one case, has been demonstrated to bind to the offending viral protein; however, how the policing role is accomplished had not been previously determined.
  • the antisense repression of several tumor associated proteins by p53 mRNA appears to be the method of control of the expression of cancer bearing proteins, and the range of tumor-related proteins repressed by p53 gene can explain the high incidence of other cancers found in families carrying a mutated p53 gene.
  • proteins from the BC53 family genes block the activity of a gene/protein located at the BRCA locus ("brca"), in those cases where a simple mutation in the p53 gene is the primary cause.
  • loss of activity of "brca” either by deletions or by mutations in the 17q/21-23 will also lead to early onset and "non-p53 related" inherited breast cancer.
  • the BC538 Phosphoprotein occur in regions of chromosome 17q close to the "brca” region, and changes in activity of 2, 4, 6, 11 have been noted in many cases of female breast cancer. However, galactosyl transferase kinase (no 7; 17q/23) , and myelo peroxidase (no 11; 17q/21.3) are located in more favorable positions. Over-expression of any one of these two proteins can be the trigger for initiation of cellular events resulting in breast cancer. Breast cancer related changes are common also in no 1, 3, 5, 8, 9 (see: I.B. "The BC538 Phosphoprotein"); these genes are located on chromosomes other than chromosome 17, in regions where mutations and deletions have been found in humans with breast cancer.
  • BC538.1 which is normally blocked by "brca”
  • BC538.1 is activated when mutations or deletions occur in "brca” .
  • mutations in p53 or mutations or deletions in the region of "brca” permit the expression of BC534 which leads to over-expression of estrogen sulfotransferase (chromosomal location not presently known) , oncogene tyrosine kinase flt' and other tyrosine kinases which initiates events leading to breast cancer or ovarian cancer.
  • BC53 family genes are likely to be the cause of ovarian cancer.
  • Ovarian tissue differs from breast tissue, and therefore factors influencing gene transcription in ovary are different from those in breast.
  • Different promoters programming transcription of the same gene in different tissues appear to be the major choice in eukaryotes for regulating the expression of the same gene in different tissues.
  • the two promoter systems ⁇ BC53/reg' which can regulate expression of BC53 family genes are consistent with expression of BC534, BC538 and BC538.1 in both breast and ovarian tissue.
  • genes and proteins discovered and described here appear to be able to escape when programmed or incidental mutations occur in crucial regions of the p53 gene.
  • the locus of these genes might be identified already as other breast cancer loci on chromosome #17; although any other localization is also feasible.
  • the sense - antisense relationship between p53 and BC534/BC538 makes the latter proteins ideal candidates for the direction of immunotherapeutic reagents to treat and cure breast cancer in women. Also, because these proteins are not expressed in healthy women, side effects from any such therapeutics should be nonexistent.
  • the diagnostic and/or therapeutic reagents of the invention additionally include fusion proteins, antipeptide reagents, etc., made from the deduced amino acid sequences described in the embodiment of Figures IB and ID.
  • the invention also includes any cellular protein activated by the proteins or domains of the proteins described in the embodiment of Figures IB, ID or IE.
  • the elucidation of the significance of BC534 or BC538 in the etiology of breast cancer provides improved means for diagnosing the presence and clinical grade of the disease. Moreover, it provides an improved means for predicting whether an asymptomatic individual is predisposed to the disease. It further provides a means for treating the disease.
  • any of the proteins described in Figures IB, ID or IF or mutants thereof may be used in the treatment of, or in the development of reagents for the treatment of, these diseases and conditions.
  • BC538 or BC538 are expressed (or expressed in significant amounts) by normal cells, the detection of these molecules in a tissue or fluid sample -- such as a biopsy sample, or a blood or lymph fluid sample -- is indicative of the presence of breast cancer in a patient.
  • antibodies are employed that are capable of binding to the products of the BC534, BC538 or BC538.1 genes, and the presence of such molecules is determined via an immunoassay.
  • suitable immunoassay formats have been described (Yolken, R.H., Rev. Infect. Pis. 4:35 (1982); Collins, W.P., In: Alternative Immunoassays, John Wiley S Sons, NY (1985); Ngo, T.T. et al.. In: Enzyme Mediated Immunoassay. Plenum Press, NY (1985) ; incorporated by reference herein.
  • Suitable antibodies can be either polyclonal or monoclonal, of either a species homologous to or heterologous to the species from which the sample was derived.
  • equivalent binding molecules such as antibody fragments (F(ab'), F(ab') 2 , single chain antibodies, etc.), recombinant antibodies, chimeric antibodies, etc. may be employed.
  • Such antibodies can be obtained using conventional methods with the gene products of either the BC534, BC538 or BC538.1 gene as an antigen. Such gene products are preferably obtained through the expression of the gene sequences described herein.
  • the simplest immunoassay involves merely incubating an anti-BC534, anti-BC538 or anti-BC538.1 antibody with a sample suspected to contain the target molecule -- the protein product of the BC534, BC538 or BC538.1 gene.
  • the presence of the target molecule is determined by the presence, and proportional to the concentration, of any antibody bound to the target molecule.
  • a solid phase is typically employed.
  • the sample can be passively bound to a solid support, and, after incubation with the antibody, the support can be washed to remove any unbound antibody.
  • the concentration of the target molecule is determined by binding the antibody to a support, and then permitting the support to be in contact with a sample suspected to contain the target molecule.
  • Target molecules that have become bound to the immobilized antibody can be detected in any of a variety of ways.
  • the support can be incubated in the presence of a labelled, second antibody that is capable of binding to a second epitope of the target molecule. Immobilization of the labelled antibody on the support thus requires the presence of the target, and is proportional to the concentration of the target in the sample.
  • the target is incubated with the sample and with a known amount of labelled target. The presence of any target molecules in the sample competes with the labelled target molecules for antibody binding sites.
  • the amount of labelled target molecules that are able to bind the antibody is inversely proportional to the concentration of target molecule in the sample.
  • radioisotopic immunoassays have the advantages of simplicity, sensitivity, and ease of use. Radioactive labels are of relatively small atomic dimension, and do not normally affect reaction kinetics. Such assays suffer, however, from the disadvantages that, due to radioisotopic decay, the reagents have a short shelf-life, require special handling and disposal, and entail the use of complex and expensive analytical equipment. RIAs are described in Laboratory Techniques and Biochemistry in Molecular Biolocry, by Work, T.S., et. al..
  • Enzyme-based immunoassay formats have the advantage that they can be conducted using inexpensive equipment, and with a myriad of different enzymes, such that a large number of detection strategies -- colorimetric, pH, gas evolution, etc. -- can be used to quantitate the assay.
  • the enzyme reagents have relatively long shelf-lives, and lack the risk of radiation contamination that attends to RIA use.
  • ELISAs are described in ELISA and Other Solid Phase Immunoassays (Kemeny, D.M. et al.
  • enzyme labels are particularly preferred. No single enzyme is ideal for use as a label in every conceivable immunometric assay. Instead, one must determine which enzyme is suitable for a particular assay system. Criteria important for the choice of enzymes are turnover number of the pure enzyme (the number of substrate molecules converted to product per enzyme site per unit of time) , purity of the enzyme preparation, sensitivity of detection of its product, ease and speed of detection of the enzyme reaction, absence of interfering factors or of enzyme-like activity in the test fluid, stability of the enzyme and its conjugate, availability and cost of the enzyme and its conjugate, and the like.
  • Suitable enzymes include peroxidase, acetylcholine esterase, alpha-glycerol phosphate dehydrogenase, alkaline phosphatase, asparaginase, ⁇ -galactosidase, catalase, delta-5-steroid isomerase, glucose oxidase, glucose-6- phosphate dehydrogenase, glucoamylase, glycoamylase, luciferase, malate dehydrogenase, peroxidase, ribonuclease, staphylococcal nuclease, triose phosphate isomerase, urease, yeast-alcohol dehydrogenase, etc.
  • Radioisotopic labels include 3 H, X11 ln , 125 I , 131 I , 32 P , 35 S , 14 C , 51 Cr , 57 To , 58 Co , 59 Fe , 7B Se ,
  • chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label, an aequorin label, etc.
  • suitable fluorescent labels include a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, an o-phthaldehyde label, a fluorescamine label, etc.
  • the presence of BC534, BC538 or BC538.1 in a cell can be determined by any means capable of detecting BC534, BC538 or BC538.1 mRNA.
  • molecules comprising nucleic acid probes capable of hybridizing to the BC534 BC538 or BC538.1 sequences of Figure 1 may be used in the diagnosis of breast cancer.
  • a "probe" is a detectably labelled nucleic acid molecule that is capable of hybridizing to a defined site of a target molecule.
  • any of the nucleotide sequences disclosed herein can be used to define a probe; the general requirement for such use being merely that the nucleic acid molecule be sufficiently long (generally 10 or more nucleotides in length) that it possesses the capacity to form stable hybridization products with the target molecule.
  • Any of a wide variety of labels may be used to label nucleic acids: enzyme labels (Kourilsky et al.. U.S. Patent 4,581,333), radioisotopic labels (Falkow et al.. U.S. Patent 4,358,535; Berninger, U.S. Patent 4,446,237), fluorescent labels (Albarella et al.. EP 144914) , chemical labels (Sheldon III et al.
  • nucleic acid based assays may use either DNA or RNA to detect the BC534, BC538 or BC538.1 mRNA.
  • the assays may be performed on RNA that has been extracted from breast cells. Alternatively, and more preferably, the assays may be done in situ on biopsied tissue.
  • BC534, BC538 or BC538.1 mRNA in a sample is too low to be detected, such mRNA may be specifically amplified through the use of any of a variety of amplification protocols, such as PCR
  • diagnosis of BC534, BC538, BC538 or BC53/reg expression is performed using a ribozyme produced from nucleic acid molecules having a sequence of SEQ ID N0:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.
  • the present invention additionally provides a capacity to predict whether an individual is at risk for breast cancer.
  • any of the above-described assays may be performed on an asymptomatic individual in order to assess that individual's predisposition to breast cancer.
  • the present invention provides a means for treating breast cancer.
  • Such treatment may be either “prophylactic” or "therapeutic.”
  • a prophylactic treatment is one that is provided in advance of any symptom of breast cancer in order to prevent or attenuate any subsequent onset of the disease.
  • a therapeutic treatment is one that is provided in response to the onset of a symptom of breast cancer, and serves to attenuate an actual symptom of the disease.
  • such treatment is provided by administering to a patient in need of such treatment an effective amount of an antibody, or an antibody fragment (F(ab'), F(ab') 2 , single chain antibodies, etc.) that is capable of binding to the product of the BC534, BC538 or BC 538.1 gene.
  • an effective amount is an amount sufficient to mediate a clinically significant change in the severity of a symptom, or a clinically significant delay in the onset of a symptom.
  • polyclonal or monoclonal antibodies may be administered. More preferably, and especially for chronic administration, the use of non- immunogenic antibodies is preferred.
  • Such molecules can be pseudo-homologous (i.e. produced by a non-human species, but altered to a form that is immunologically indistinct from human antibodies) . Examples of such pseudo-homologous molecules include "humanized" (i.e. non-immunogenic in a human) prepared by recombinant or other technology.
  • Such antibodies are the equivalents of the monoclonal and polyclonal antibodies, but are less immunogenic, and are better tolerated by the patient.
  • Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e. chimeric antibodies) (Robinson, R.R. et al. , International Patent Publication PCT/US86/02269; Akira, K. et al.. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison, S.L. et al.. European Patent Application 173,494; Neuberger, M.S. et al.. PCT Application WO 86/01533; Cabilly, S. et al. , European Patent Application 125,023; Better, M. et al..
  • Suitable "humanized” antibodies can alternatively be produced by CDR or CEA substitution (Jones, P.T. et al.. Nature 321:552-525 (1986) ; Verhoeyan et al.. Science 239:1534 (1988); Beidler, C.B. et al.. £».
  • nucleic acid molecules of the present invention may be mutated and expressed in order to identify BC534, BC538 or BC538.1 mutant gene products that can complex with and significantly inactivate normal BC534, BC538 or BC538.1 gene products present in a tumor cell.
  • such mutated protein molecules may be administered to a patient.
  • nucleic acid expressing such molecules may be administered.
  • "antisense" or "triplex" nucleic acid molecules may be used to provide the desired therapy.
  • an "antisense oligonucleotide” is a nucleic acid (either DNA or RNA) whose sequence is complementary to the sequence of at least part of the BC534, BC538, BC538.1 the protein- encoding sequences or the regulatory sequences in BC53/reg described herein, such that it is capable of binding to, or hybridizing with, an endogenous BC534, BC538 or BC538.1 mRNA molecule, and can thereby impair (i.e. attenuate or prevent) its the translation into BC534, BC538 or BC538.1 gene products.
  • a "triplex" molecule is a nucleic acid molecule that is capable of binding to double-stranded DNA in a manner sufficient to impair its transcription.
  • the nucleic acid molecule must be capable of binding to the BC534, BC538, or BC538 gene sequences of the double-stranded DNA genome in a manner sufficient to impair the transcription of either gene.
  • Triplex oligonucleotides are disclosed by Hogan, U.S. Patent 5,176,996 and by Varma et al.. U.S. Patent 5,175,266.
  • the nucleic acid molecule must be capable of binding to or hybridizing with that portion of the BC534, BC538 or BC538.1 mRNA molecule which mediates the translation of the target mRNA; or BC53/reg which programs transcription of their mRNAs.
  • Antisense oligonucleotides are disclosed in European Patent Application Publication Nos. 263,740; 335,451; and 329,882, in U.S. Patent 5,097,617 and in PCT
  • an antisense oligonucleotide that is designed to specifically block translation of a BC534 or BC538 mRNA transcript can be used to impair the expression of the BC534 or BC538 genes in a cell, and thereby provide a treatment for breast cancer.
  • the antisense oligomer is prepared in accordance with the nucleotide sequence of BC534 or BC538 as reported herein.
  • the sequence of the antisense oligonucleotide may contain one or more insertions, substitutions, or deletions of one or more nucleotides provided that the resulting oligonucleotide is capable of binding to or hybridizing with the above-described translation locus of either BC534, BC538 or BC538.1 mRNA.
  • any means known in the art to synthesize the antisense oligonucleotides of the present invention may be used (Zamechik et al.. Proc. Natl. Acad. Sci. (U.S.A.) £1:4143 (1986); Goodchild et al.. Proc. Natl. Acad. Sci. (U.S.A.) 85:5507 (1988); Wickstrom et al.. Proc. Natl. Acad. Sci. (U.S.A.) £5:1028; Holt, J.T. et al. , Molec. Cell. Biol. 8:963 (1988); Gerwirtz, A.M. et al.
  • the antisense oligonucleotides of the present invention may be prepared using solid phase "phosphoramidite synthesis.”
  • the synthesis is performed with the growing nucleotide chain attached to a solid support derivatized with the nucleotide which will be the 3'-hydroxyl end of the oligonucleotide.
  • the method involves the cyclical synthesis of DNA using monomer units whose 5'-hydroxyl group is blocked (preferably with a 5' -DMT (dimethoxytrityl) group), and whose amino groups are blocked with either a benzoyl group (for the amino groups of cytosine and adenosine) or an isobutyryl group (to protect guanosine) .
  • Methods for producing such derivatives are well known in the art.
  • compositions can be formulated according to known methods used to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle.
  • Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980)).
  • compositions will contain an effective amount of such agents, together with a suitable amount of carrier vehicle.
  • Control release preparations may be achieved through the use of polymers to complex or absorb the agents.
  • the controlled delivery may be exercised by selecting appropriate macromolecules (for example polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methyl- cellulose, carboxymethylcellulose, or protamine, sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release.
  • Another possible method to control the duration of action by controlled release preparations is to incorporate the agents into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copoly ers.
  • microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatine-microcapsules and poly(methylmethacylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.
  • nucleic acid molecule(s) comprising antisense or triplex molecules, or encoding mutated BC534, BC538 or BC538.1 gene products may be administered using viral or retroviral vectors in accordance with the methods of "gene therapy” .
  • the principles of gene therapy are disclosed by
  • the BC534 and BC538 genes were prepared by oligonucleotide synthesis using solid phase gene assembly. Successive oligonucleotides phosphorylated and added at molar excess were attached stepwise to a growing chain anchored to a solid phase support. The assembled oligonucleotides were ligated with T4 DNA ligase and detached from the support by cleaving at an added restriction enzyme site. The constructed genes were processed in the EXPRESS system (Invitrogen Corp.: pTrcHis Xpress-Prokaryotic Expression and Purification system) according to the manufacturer's instructions. In this manner, two DNA molecules, encoding proteins of 21 and 44 amino acids, respectively, were constructed.
  • EXPRESS system Invitrogen Corp.: pTrcHis Xpress-Prokaryotic Expression and Purification system
  • nucleic acid sequences of these molecules are 100% homologous to regions on the antisense strand of human p53 gene.
  • the expression of these proteins is repressed by the normal wild type p53 gene healthy in women; however, mutations, inherited or acquired, in regions of the p53 gene which are crucial for maintaining repression could permit the expression of various levels of these two antisense proteins.
  • the antisense proteins are potential intracellular activators of tumor causing and tumor associated proteins and of an inhibitor of 17 ⁇ hydroxy steroid interaction with its receptor.
  • the antisense relationship with p53 gene and the functions of these proteins make them ideal candidates for causative factors of p53 related female breast cancer and some other cancers which correlate with this disease in families carrying the mutated p53 gene. Since these proteins are not expressed (or expressed at insignificant levels) in normal humans they are excellent targets for developing humanized antibodies against to prevent and cure the disease.
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE cDNA
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • ORIGINAL SOURCE

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Abstract

Agents et procédés destinés au diagnostic et au traitement du cancer du sein et des pathologies connexes. Ces agents comprennent des molécules antisens de trois gènes impliqués dans le cancer du sein, et des éléments promoteurs régulant la transcription de ces gènes, ainsi que leurs analogues et dérivés, et des molécules protéiques exprimées par ces gènes.
PCT/EP1994/000651 1993-03-16 1994-03-04 Agents de prevention et de traitement du cancer du sein WO1994021791A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015662A3 (fr) * 1995-10-26 1997-07-10 Ribozyme Pharm Inc Procede et reactif de traitement de maladies ou troubles lies aux niveaux du recepteur du facteur de croissance endotheliale vasculaire
WO1998007851A3 (fr) * 1996-08-22 1998-08-13 Johanna E Bergmann Agents permettant la detection, la prevention, et le traitement, avant apparition des symptômes, du cancer du sein chez les humains
WO1998055650A1 (fr) * 1997-06-04 1998-12-10 Rijksuniversiteit Te Leiden Trousse de controle diagnostique servant a determiner une predisposition au cancer du sein et des ovaires, materiaux et procedes servant a effectuer cette determination
EP1408111A3 (fr) * 1995-10-26 2004-09-08 Ribozyme Pharmaceuticals, Inc. Procédé et réactif de traitement de maladies ou troubles liés aux niveaux du récepteur du facteur de croissance endothéliale vasculaire
US7034009B2 (en) 1995-10-26 2006-04-25 Sirna Therapeutics, Inc. Enzymatic nucleic acid-mediated treatment of ocular diseases or conditions related to levels of vascular endothelial growth factor receptor (VEGF-R)
US7176304B2 (en) 2002-02-20 2007-02-13 Mcswiggen James RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US7517864B2 (en) 2001-05-18 2009-04-14 Sirna Therapeutics, Inc. RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)

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WO1990009180A1 (fr) * 1989-02-15 1990-08-23 Board Of Regents, The University Of Texas System Procedes et compositions de traitement du cancer a l'aide d'oligonucleotides

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Title
BERGMANN J, ET AL.: "A PROTEIN DRP90 ENCODED ON THE LEFTWARDS STRAND OF SOYBEAN NODULE URATE OXIDASE cDNA BINDS TO A REGULATORY SEQUENCE IN LEGHEMOGLOBIN C-3 GENE.", NUCLEIC ACIDS RES 19 (6). 1991. 1338 *
HARLOW, E. ET AL.: "Molecular cloning and in vitro expression of a cDNA clone for human cellular tumor antigen p53", MOLECULAR AND CELLULAR BIOLOGY, vol. 5, no. 7, July 1985 (1985-07-01), WASHINGTON US, pages 1601 - 1610 *
MERINO, E. ET AL.: "Antisense overlapping open reading frames in genes from bacteria to humans", NUCLEIC ACIDS RESEARCH, vol. 22, no. 10, May 1994 (1994-05-01), ARLINGTON, VIRGINIA US, pages 1903 - 1908 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997015662A3 (fr) * 1995-10-26 1997-07-10 Ribozyme Pharm Inc Procede et reactif de traitement de maladies ou troubles lies aux niveaux du recepteur du facteur de croissance endotheliale vasculaire
US6566127B1 (en) 1995-10-26 2003-05-20 Ribozyme Pharmaceuticals, Inc. Method and reagent for the treatment of diseases or conditions related to levels of vascular endothelial growth factor receptor
EP1408111A3 (fr) * 1995-10-26 2004-09-08 Ribozyme Pharmaceuticals, Inc. Procédé et réactif de traitement de maladies ou troubles liés aux niveaux du récepteur du facteur de croissance endothéliale vasculaire
US6818447B1 (en) 1995-10-26 2004-11-16 Sirna Therapeutics, Inc. Method and reagent for the treatment of diseases or conditions related to levels of vascular endothelial growth factor receptor
US7034009B2 (en) 1995-10-26 2006-04-25 Sirna Therapeutics, Inc. Enzymatic nucleic acid-mediated treatment of ocular diseases or conditions related to levels of vascular endothelial growth factor receptor (VEGF-R)
WO1998007851A3 (fr) * 1996-08-22 1998-08-13 Johanna E Bergmann Agents permettant la detection, la prevention, et le traitement, avant apparition des symptômes, du cancer du sein chez les humains
WO1998055650A1 (fr) * 1997-06-04 1998-12-10 Rijksuniversiteit Te Leiden Trousse de controle diagnostique servant a determiner une predisposition au cancer du sein et des ovaires, materiaux et procedes servant a effectuer cette determination
US6733966B1 (en) 1997-06-04 2004-05-11 Rijksuniversiteit Te Leiden Diagnostic test kit for determining a predisposition for breast and ovarian cancer, materials and methods for such determination
US7507800B2 (en) 1997-06-04 2009-03-24 Rijksuniversiteit Te Leiden Diagnostic test kit for determining a predisposition for breast and ovarian cancer, materials and methods for such determination
US7517864B2 (en) 2001-05-18 2009-04-14 Sirna Therapeutics, Inc. RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)
US7176304B2 (en) 2002-02-20 2007-02-13 Mcswiggen James RNA interference mediated inhibition of vascular endothelial growth factor and vascular endothelial growth factor receptor gene expression using short interfering nucleic acid (siNA)

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