WO1999009047A1 - Detection et traitement du cancer du sein, des cancers gynecologiques et du cancer de la prostate - Google Patents

Detection et traitement du cancer du sein, des cancers gynecologiques et du cancer de la prostate Download PDF

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WO1999009047A1
WO1999009047A1 PCT/US1998/015580 US9815580W WO9909047A1 WO 1999009047 A1 WO1999009047 A1 WO 1999009047A1 US 9815580 W US9815580 W US 9815580W WO 9909047 A1 WO9909047 A1 WO 9909047A1
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hiv
cancer
mab
breast
cells
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PCT/US1998/015580
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Eva M. Rakowicz-Szulczynska
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Rakowicz Szulczynska Eva M
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Priority to EP98938042A priority Critical patent/EP1012159A1/fr
Priority to CA002297727A priority patent/CA2297727A1/fr
Priority to AU86654/98A priority patent/AU8665498A/en
Publication of WO1999009047A1 publication Critical patent/WO1999009047A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1045—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against animal or human tumor cells or tumor cell determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • 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
    • G01N33/57411—Specifically defined cancers of cervix
    • 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
    • G01N33/57415—Specifically defined cancers of breast
    • 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
    • G01N33/57434—Specifically defined cancers of prostate
    • 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
    • G01N33/57442—Specifically defined cancers of the uterus and endometrial
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/77—Internalization into the cell
    • 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
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/15—Retroviridae, e.g. bovine leukaemia virus, feline leukaemia virus, feline leukaemia virus, human T-cell leukaemia-lymphoma virus
    • G01N2333/155—Lentiviridae, e.g. visna-maedi virus, equine infectious virus, FIV, SIV
    • G01N2333/16—HIV-1, HIV-2

Definitions

  • the present invention relates to newly discovered polypeptides associated with breast cancer, gynecological cancer, and prostate cancer, to DNA molecules encoding the polypeptides, and to immunoassay methods for detecting these antigens in biological samples, as well as immunotherapeutic methods for treating these cancers with antibodies that bind to these antigens. More specifically, the invention relates to the discovery of immunological cross-reactivity between antibodies to Human Immunodeficiency Virus (HIV-1) proteins and certain polypeptides in breast, gynecological, and prostate carcinomas. This cross-reactivity results in the formation of new immunocomplexes which are useful in the immunodiagnostic methods of this invention.
  • HIV-1 Human Immunodeficiency Virus
  • MAb monoclonal antibodies
  • direct approaches MAb IgG2a and IgG3 mediate antibody-dependent cellular cytotoxicity and/or exert complement-dependent cytotoxicity.
  • radio-immunotherapy with radioactively labeled MAb.
  • Immunotoxins which are the conjugates of MAb with the subunit A of the ricin or diphtheria toxin, exhibit high tumoricidal potential, however have a restricted application due to high cytotoxicity.
  • the present invention provides novel polypeptides and DNA sequences, which are associated with breast, gynecological, and prostate carcinomas but not with normal cells.
  • the present invention also provides methods for detecting and treating breast, gynecological, and prostate cancers.
  • these methods utilize antibodies developed against a polypeptide corresponding to regions in the gpl20, p41 and gag proteins of Human Immunodeficiency Virus (HTV-l).
  • an antibody used in the methods of this invention is directed against the first 204 amino acids of gpl20, amino acids encoded by nucleotides 1151-1665 of the gag region, amino acids encoded by nucleotides 7801-7820 of the gp41 region of HIV- 1.
  • the antibody is a monoclonal antibody developed against a synthetic peptide corresponding to the variable domain of HIV- 1 gpl20 (amino acid region 308-322).
  • the monoclonal antibody is MAb 5023 (ATCC NO. HB 10043), which can be purchased from DuPont/NEN and is listed in the catalog of this company entitled “DuPont/NEN Research Products 1992-1993" under Catalog Number, NEA-9305, the Product description of which is "HIV Monoclonal Antibody gpl20-Neutralizing (sequence specific) (mouse) 0.5mL".
  • MAb 5023 has been found to be unique in its ability to penetrate the cell and localize within the cell nucleus and in its ability to recognize a family of antigens expressed by breast cancer and gynecological cancers.
  • this invention makes possible a simple and direct in vivo diagnosis of cervical cancer by administration of MAb 5023 to the cervix and then detecting the presence of immunocomplexes formed between said antibody and cell surface proteins pl20 and p41 is another embodiment.
  • the method comprises detecting HIV-I-crossreactive breast carcinoma-associated antigens in a biological sample comprising (a) exposing a biological sample, suspected of containing the antigens, to an antibody which recognizes the following cell membrane proteins: pl60, p80, p45, and p24 and the following chromatin protein: p24, and (b) detecting the presence of immunocomplexes formed between said antibody and said proteins.
  • a method for detecting HIV-I-crossreactive, gynecological cancer-associated antigens in a biological sample comprising (a) exposing a biological sample, suspected of containing the antigens, to an antibody which recognizes the following cell membrane proteins, pl20, p41, and p24 and the chromatin protein, p24, and (b) detecting the presence of immunocomplexes formed between said antibody and said proteins.
  • the immunocomplexes thus formed constitute an embodiment of this invention and comprise (a) at least one of the following cell membrane proteins, pl60, p80, p45, or p24, or the chromatin protein, p24, and (b) an antibody which recognizes each of said proteins.
  • the antibody is MAb 5023.
  • the immunocomplexes of this invention are in an immobilized form. In this form the immunocomplexes are insolubilized, or otherwise supported, on a variety of standard immobilization substrates. Examples of materials to which the capturing agents can be attached are glass, synthetic polymers, synthetic resins, cellulose, nitrocellulose, and various metals.
  • Another aspect of the present invention is a method for treating breast cancer and gynecological cancer, selected from the group consisting of ovarian, cervical, endometrial, and vulvar cancer, comprising administering to a patient an antibody which translocates and internalizes to the nucleus of the cell and which forms immunocomplexes with the HIV crossreactive antigens of this invention, e.g., MAb 5023.
  • the antibody can be administered alone or conjugated with a radioactive or cytotoxic ligand, and is administered in a pharmaceutically effective dosage form, generally, for intravenous or intraperitoneal injection.
  • the HIV-I-crossreactive cancer-associated antigens of this invention are the pl60, p80, p45, and p24 cell membrane proteins and the p24 chromatin protein for breast cancer, and pl20, p41, and p24 cell membrane proteins and the p24 chromatin protein for gynecological cancers (i.e., ovarian, cervical, endometrial , and vulvar cancer ) .
  • the antibody may be labeled with an enzyme, fluorochrome, radioisotope, or luminescer.
  • the step for detection would normally be by enzyme reaction, fluorescence, radioactivity, or luminescence emissions, respectively.
  • Bodily secretions, bodily fluids, and tissue specimens are all suitable samples in this regard.
  • bodily secretions include cervical secretions, vaginal secretions, human breast milk, urine, and intraperitoneal a ⁇ citic fluid.
  • Suitable bodily fluids include blood.
  • tissue specimens include a variety of biopsies, such as a biopsy of cervical dysplasia, cervical cancer, ovarian cancer, vulvar cancer, lymph nodes, and bone marrow.
  • tissue from all areas may be examined in connection with a post mortem examination, including primary tumors and metastatic tumors , lymph nodes, bone marrow and all organs .
  • continuous hybrid cell lines can be established that produce monoclonal antibodies directed against an antigenic determinant of the HIV-I-cro ⁇ reactive cancer-associated antigens of this invention for use in the methods described above.
  • the cell line comprises the hybridoma clone for MAb 5023.
  • FIGURES 1A-1E Immunofluorescence staining of SKBr 5
  • FIGURE 2 Autoradiographic detection of free 125 I-MAb 5023 (lane 1) and 125 I-MAb 5023 accumulated in the cytoplasm ( lane 2 ) and in the chromatin ( lane 3) after 24 h incubation with SKBr5 cells. Cytoplasm and chromatin were prepared from 5 x 10 5 cells.
  • FIGURES 3A and 3B Western blotting of different MAb against V3 loop of HIV-I gpl20 with membrane (M) and chromatin (Ch) proteins of SKBr5 cells separated in a 7.5% (A) or 13% (B) polyacrylamide gel.
  • FIGURES 4A and 4B Western blotting of MAb 5023 with membrane (M) and chromatin (Ch) proteins of different cell lines separated in 7.5% (A) or 13% (B) polyacrylamide gel.
  • FIGURE 5 Electrophoretic analysis (7.5% polyacrylamide gel ) and autoradiographic detection of [ 35 S]methionine-labeled plasma membrane protein, immunoprecipitated by MAb 5023 from SKBr5 breast carcinoma cells (lane 1) and SW707 colorectal carcinoma cells (lane 2).
  • FIGURE 6 Western blotting of human HIV-I- neutralizing antibodies to env-2-3 with SKBr5 breast carcinoma membrane proteins (lane 1) and HIV-I gpl20 (lane 2). Proteins are separated in 7.5% polyacrylamide gel.
  • FIGURES 7A-7D Western blotting of MAb 5023 (7A, 7B, 7C) and of MAb 5025 (7D) against HIV-I gpl20 with plasmamembrane-containing cytoplasm (C) or nuclear fraction (N), or pure chromatin (Ch). Proteins were separated in 7.5-10% polyacrylamide gel. Different migration is due to the different time of electrophoresis and different gel concentrations. Prestained protein markers (high and low molecular weight from Bio-Rad) were used in each experiment.
  • FIGURE 8 Effect of MAb 5023 on growth of cervical cancer cell line SiHa.
  • the inhibitory concentration 0.25 corresponds to 0.05 ⁇ g/ml.
  • FIGURES 9A-9C Electrophoretic analysis (10% gel) and Western blotting of cytoplasm (C) chromatin (Ch), or total (C+N) proteins from cancer (A and B, lane 2) and normal cells (B, lane 2 and C) with MAb anti HIV-I gpl20 (Cells or sections of cancer or normal tissues, obtained during standard surgical procedures, were homogenized in 0.35 M sucrose/10 mM KC1/1.5 mM MgCl2/10 mM Tris-HCl (pH 7.6)/0.12% Triton X-100/12 mM 2-mercaptoethanol (1 ml/3 X 10 6 cells or 0.25 - 1 ml/cm tissue), and centrifuged at 600 x g for 10 minutes.
  • MAb anti HIV-I gpl20 Cells or sections of cancer or normal tissues, obtained during standard surgical procedures, were homogenized in 0.35 M sucrose/10 mM KC1/1.5 mM MgCl2/10 mM Tris-HC
  • Supernatant was the crude, membrane-containing cytoplasmic fraction.
  • the nuclear pellet was first washed with 0.2 M sucrose/3 mM CaCl2/50 mM Tris-HCl (pH 7.6), and then with 0.14 M NaCl/10 mM Tris-HCl (pH 8.3) and centrifuged at 700 x g for 10 minutes.
  • the pellet was swollen in 1 mM Tris-HCl (pH 7.9) and centrifuged throughout the 1.7 M sucrose 10 mM Tris HCl (pH 7) at 160,000 x g for 80 minutes. Chromatin was pelleted at the bottom of the tube.
  • Proteins were separated in 10% polyacrylamide gel with 0.1% sodium dodecyl sulfate (SDS) in 250mM Tris-HCl (pH 8.3), 195mM glycine, and 0.1% SDS, according to Laemmli et al (76). Each lane corresponds to 3 X 10 5 , except lane 7 which corresponds to 5 X 10 4 cells. Blotting of proteins from the polyacrylamide gel to the PVDF membrane was performed in 25mM Tris-HCl (pH 8.6), 192mM glycine buffer, containing 10% methanol.
  • SDS sodium dodecyl sulfate
  • Filters were incubated with 1% BSA for 16 h, at 0°C, then with MAb anti-HIV-I gpl20 (DuPont ) ( 5 ⁇ g/ml ) , washed with Tris-glycine buffer, and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG for 1 h. After washing with TBST, membranes were incubated with 0.1% 1-naphthyl-phosphate and Fast Red, in lOOmM Tris-HCl pH 9.5, lOOmM NaCl, 5mM MgCl2).
  • A MCF7 breast carcinoma (lanes 1,2), SiHa cervical carcinoma (lanes 3,4), endometrial cancer (lanes 5,6) and cervical cancer (lane 7) proteins.
  • B ovarian cancer (lane 1) and normal ovarian tissue (lane 2).
  • C normal skin, muscle and cervical tissue and normal vaginal mucosal proteins.
  • FIGURE 10 Competitive inhibition of MAb anti HIV-I gpl20 binding to the cytoplasmic proteins extracted from mixed Mullerian tumor (A) by peptide RIQRGPGRAFVTIGK SEQ ID NO:l (B) , against which the MAb was developed. The same amount of proteins were separated in A and B, lane 1 (equivalent of 10 cells) and in A and B, lane 2 (equivalent of 0.5 X 10 ) (Peptide (18 ⁇ g/ml) was incubated with MAb anti- HIV-I gpl20 (5 ⁇ g/ml) for 1 hr on ice, next it was incubated with the filter (B). In control (A), the filter was incubated with MAb alone).
  • the nuclear pellet was first washed with 0.2 M sucrose/3 mM CaCl2/50 mM Tris-HCl (pH 7.6), and then with 0.14 M NaCl/10 mM Tris-HCl (pH 8.3) and centrifuged at 700 x g for 10 minutes.
  • the pellet was swollen in 1 mM Tris-HCl (pH 7.9) and centrifuged throughout the 1.7 M sucrose 10 mM Tris HCl (pH 7) at 160,000 x g for 80 minutes. Chromatin was pelleted at the bottom of the tube.
  • Proteins were separated in 10% polyacrylamide gel with 0.1% sodium dodecyl sulfate (SDS) in 250mM Tris-HCl (pH 8.3), 195mM glycine, and 0.1% SDS, according to Laem li et al (76). Each lane corresponds to 3 X 10 , except lane 7 which corresponds to 5 X 10 4 cells. Blotting of proteins from the polyacrylamide gel to the PVDF membrane was performed in 25mM Tris-HCl (pH 8.6), 192mM glycine buffer, containing 10% methanol.
  • SDS sodium dodecyl sulfate
  • Filters were incubated with 1% BSA for 16 h, at 0°C, then with MAb anti-HIV-I gpl20 (DuPont) (5 ⁇ g/ml) , washed with Tris-glycine buffer, and incubated with alkaline phosphatase-conjugated goat anti-mouse IgG for 1 h. After washing with TBST, membranes were incubated with 0.1% 1-naphthyl-phosphate and Fast Red, in lOOmM Tris-HCl pH 9.5, lOOmM NaCl, 5mM MgCl2).
  • FIGURES 11A and 11B Representative DNA amplification analysis with primers SK 68/SK 69 (A) and with P1/P2 (B) primers (Polymerase chain reaction (PCR) occurred in the solution containing: lOmM KC1, lOmM (NH4)SO__, 200mM Tris-HCl, 20mM MgS ⁇ 4 , 1% Triton X-100 dNTP (lOmM each of dATP, dTTP, dCTP, and dGTP), 120-250ng of the primer, 2.5U Taq polymerase, water up to 30 ⁇ l. DNA template was added at the amount of 0.2-l ⁇ g (20 ⁇ l), and paraffin oil (70-100 ⁇ l). The reaction occurred in a thermal cylinder (30-40 cycles). PCR. products were separated in 1% agarose gel with ethidium bromide).
  • lane 1 123bp marker
  • lane 2 HIV-I infected T-cells
  • 3 SiHa
  • 4 MCF7
  • 5 mixed Mullerian tumor
  • 6-8 normal skin (different patients)
  • 9 no template
  • 10 endometrial cancer
  • B) lane 1 HIV-I infected T-cells, 2-3: SiHa (1 ⁇ g and 0.1 ⁇ g, respectively), 4: mixed Mullerian tumor, 5-6: MCF7 (1 ⁇ and 0.1 ⁇ g, respectively), 7: endometrial cancer, 8: normal skin, 9: 123bp marker
  • FIGURE 12 Sequences of 140-150bp DNA fragments of cervical cancer SiHa, endometrial cancer obtained during standard surgery (EIV) and breast cancer MCF7 amplified by PCR with primers SK 68/SK 69 (see Fig. 3A) . Nucleotides identical in at least two or more cell types are boxed. 21bp of the region identical or with high homology to HIV-I are marked with stars.
  • FIGURES 13A-13E Transmission electron micrograph of viral particles in SiHa (A,C) and MCF7 (B,C) cells and of extracellular vesicle obtained from MCF7 cells (D) . Viral particles negatively stained with uranyl acetate (E). In A, D and E, samples were immuno-gold labeled with MAb anti-HIV-I gpl20. The sizes of the immuno-gold particles were 15nm (A,D) and lOnm (E). V - viral particles
  • FIGURES 14A and 14B Effect of antisense oligonucleotide RAK-I on growth of breast cancer MCF7 cell line.
  • Cells were grown for 4 days in the absence (A) or presence (B) of the antisense oligonucleotide RAK-I (5'-CCAGACTGTGAGTTGCAACAG-3') SEQ ID NO:6 added daily at the concentration of lOO ⁇ g/ml (day 1) and 50 ⁇ g/ml (days 2 and 3).
  • Oligonucleotide 5 -TGTGACATCAGGCTCAAATC-3' was used as a negative control and did not affect cell growth (not shown).
  • FIGURE 15 Breast Cancer Sequences from patients. DNA sequences from breast cancer tissue of seven patients (1-7) were amplified by using SK68/SK69 primers. The PCR products were sequenced and compared with each other and with PCR product from HIV-1 infected cells. The dotted lines indicate the location of the primers. Sequences noted by a dash are variable.
  • PCR conditions PCR occurred in the solution containing: lOmM KC1, lOmM (NH 4 )S0 4 , 20 mM Tris-HCl, 5mM MgCl 2 , 0.1% Triton X-100, 0.2mM dNTPs (dATP, dTTP, dCTP, and dGTP), 0.25 ⁇ m each primer and 2.5 U Taq polymerase.
  • the amount of DNA template used was l.O ⁇ g per 50 ⁇ l of mineral oil and incubated at 94°C for 4 min., 66°C for 30 seconds, 72°C for 2 min. (30 cycles). The reactions occurred in a Perkin Elmer Gene Amp 9600 thermal cycler. PCR was done using a set of HIV-1 derived primers: SK68 and SK69 primers, i.e., SEQ ED NOs: 2 and 3, respectively.
  • FIGURE 16 Prostate Cancer Sequences from patients. DNA sequences from prostate cancer tissue of six patients (1-6) were amplified by using SK68/SK69 primers. The PCR products were sequenced and compared with each other and with PCR product from HTV-1 infected cells. The dotted lines indicate the location of the primers. Sequences noted by a dash are variable in at least 2 patients.
  • One embodiment of this invention is a set of novel polypeptides and DNA sequences found in breast, gynecological, and prostate cancer cells but not in normal breast epithelial cells, melanoma cells, colorectal carcinoma cells, and non- HIV-1 infected lymphocytes.
  • MAb 5023 developed against amino acid residues 308-322 of the variable V3 loop of HIV-I, reacted in Western Blotting with a 160,00 M2 (pi 60) and 80,000 M2 (p80) cell surface antigens. Protein p 160 seems to represent an ologomeric form of p80.
  • Another MAb, 5025 directed against the same region 308-322, and Mab against amino acid regions 307-328 and 308-332, did not recognize pl60 or p80.
  • MAb 5023 and MAb 5025 were developed against the same synthetic peptide, there are minor differences in the structure of the core epitope recognized by these MAb, which account for the several-fold higher affinity of MAb 5023 than that of MAb 5025. All of the MAb tested recognized another cell membrane antigen, p45.
  • antigens pl60, pl20, p80, p45, p42, and p24 have been detected in breast cancer, and three antigens, pi 20, p41, and p24 are commonly expressed by cervical, ovarian, endometrial, and vulvar cancer. All antigens, except for p24 are selectively expressed in the cytoplasmic/plasmamembrane fraction. Protein p24 was detected in both plasmamembrane and chromatic fractions.
  • a monoclonal antibody derived by immunizing a mouse with cellular fractions from breast cancer cells was also able to recognize polypeptides associated with gynecological antigens (Rakowicz-Szulcynska, EM et al., "Nuclear Antigens With Epitopic Homology to the Cell Surface Antigens: The Neglected Determinants of Malignant Transformation," Antibodies. Immunoconjugates. and Radiopharmaceuticals 8:261-279 (1995) inco ⁇ orated herein).
  • the same antibody was able to recognize pi 20, p42, and p25 polypeptides associated with prostate cancer.
  • Proteins pl60/p80, pi 20, and p41 were undetectable in melanoma, colorectal carcinoma, normal breast epithelial cells and, in non-infected lymphocytes.
  • the low molecular weight protein p24 is expressed in epithelial cells, lymphocytes, and several cancer types.
  • Mab 5023 Internalization of Mab 5023 was also detected in cervical cancer cells which express pi 20.
  • the epitope recognized by Mab 5023 which may be involved in internalization, is well characterized.
  • the Mab 5023 was developed against amino acid sequences 308-322 (RIQRGPGRAFVTIGK) of the variable loop of HIV-I gpl20, but this Mab binds to the epitope GRAF. G preceding RAF is believed to be critical for internalization. Only Mab 5023, but none of other MAbs against HIV crossreact with cancer antigens which suggests that pi 60 and pi 20 are the proteins which mediate internalization.
  • Mab 5023 when internalized and translocated to the nucleus, stimulated RNA synthesis and promoted growth of breast cancer cells. In contrast, growth of cervical cancer cells was by 30-50% inhibited by Mab 5023.
  • MAb 5023 In addition to the potentially diagnostic and prognostic value of HIV-crossreactive cancer antigens, an ability to internalize MAb 5023 makes these antigens excellent targets for immunotherapy. Specificity of MAb 5023 interaction with breast cancer and gynecological cancer as well as internalization and chromatin binding, make MAb 5023 a potentially useful vehicle for different drugs destined to the cytoplasm and/or nucleus. In accordance with this invention, MAb 5023 is also used as a vehicle for the radioactive ligand ( 125 I) and for the antisense oligonucleotide complementary to proto-oncogene Neu/HER-2, also called erbB-2. Neu-oncogene encodes cell surface receptor pl85Neu which exhibits strong structural homology to EGF receptor.
  • radioactive ligand 125 I
  • antisense oligonucleotide complementary to proto-oncogene Neu/HER-2 also called erbB-2.
  • Neu-oncogene encodes cell surface receptor pl85
  • the oncogenic potential of Neu is released by multiple genetic mechanisms, including point mutation within the transmembrane region, truncations of noncatalytic sequences at both the cytoplasmic and the extracellular domains or by amplification. Particularly, a strong association between Neu amplification and overexpression and clinical outcome has been reported in breast and ovarian cancer. It was shown that MAbs directed against the product of the oncogene Neu inhibit growth of cancer. Down regulation of the cell surface receptor Neu expression by antisense oligonucleotides will significantly inhibit growth of breast cancer and ovarian cancer. Since MAb 5023 is efficiently internalized, another embodiment of this invention is antisense therapy, in which an oligonucleotide complementary to specific regions of the protooncogene Neu is conjugated with MAb 5023.
  • Antisense research has been expanded during the last five years. Antisense is the term coined to describe the interaction between oligonucleotides complementary to sense (pre-mRNA or mRNA) molecules that inhibit the production of the protein product. It has been broadened to describe any therapeutic oligonucleotide interaction with nucleic acids.
  • the mechanisms which are involved in antisense oligonucleotides action involve: inhibition of translation through the blocking protein binding to specific regions of mRNA, formation of the triplex between double stranded mRNA and the antisense which is degraded by the RNase H, transcriptional arrests after binding of the oligonucleotide to DNA which prevents initiation or elongation of transcription, inhibition of splicing, disruption of necessary RNA structure, destabilization, inhibition of polyadenylation and others.
  • Cholesterol, poly(L- lysine), interleukin, 1 , 2-di-O-hexadecyl-ra ⁇ - glycero-3-H-phosphate conjugated oligonucleotides have been synthesized for the purpose of utilizing specific protein-mediated endocytic pathways. Uptake of the oligonucleotides conjugated with the specific ligand was significantly higher. Mechanism of release of oligonucleotides from endosomal vesicles is not clear and it is speculated that it may be induced by conformational changes of endosomal membranes, transient membrane destabilization or other mechanisms.
  • Another embodiment of this invention is the application of a MAb which is able to internalize and localize into the cell nucleus, e.g., MAb 5023, to deliver an antisense oligonucleotide to the target cells (e.g., anti-Neu and other targets).
  • a MAb which specifically interacts with breast cancer and gynecological cancer cells provides a new opportunity to target antisense oligonucleotide into a specific locus.
  • MAb since MAb is found inside the cell in a nondegraded form, and it passes the endocytic membrane very efficiently, immuno- antisense therapy may represent an approach to more general methods of genetic therapy.
  • the MAb is specific for a peptide corresponding to the variable domain of the Human Immunodeficiency Virus (HIV-I) envelope protein gpl20 (amino acid region 308-322).
  • HIV-I Human Immunodeficiency Virus
  • animal hosts are immunized according to a conventional protocol in order to induce the development of specifically immune lymphocytes (known as plasma cells) which produce antibodies to the antigen.
  • lymphocytes are recovered from the spleen of the immunized host and are fused according to conventional experimental protocols with myeloma tumor cells derived from the same animal species to form giant somatic cell hybrids.
  • These hybrid fusion protocols originally reported by G Kohler and C Milstein .Nature 256: 495-497, 1975) are generally known by those skilled in the art.
  • the cell-cell hybrids exhibit characteristics of both parent cell types used in the fusion: like the malignant myeloma parent, fused cell hybrids have the capacity to grow rapidly and indefinitely in tissue culture; in addition, they have the capacity to secrete large amounts of the antibody specified by the genes of the normal antibody- secreting lymphocyte parent that participated in the fusion.
  • These hybrid cell lines are called "hybridoma ⁇ . " After appropriate selection and cloning, they are propagated in tissue culture or in a genetically identical or immunocompromised animal for an indefinite period in order to continuously produce antibody to the antigen.
  • the antibodies of the present invention can be labeled with any of a variety of standard substances which include radioactive, O 99/09047
  • fluorescence, or enzyme markers examples of such standard markers are:
  • Radioactive tritium, carbon-14, phosphorus 32, iodine-125;
  • Fluorescent fluorescein, rhodamine, phycoerythrin, Texas red;
  • Enzyme Horseradish peroxidase, alkaline phosphatase, B-galactosidase.
  • Cell line SiHa (cervical cancer) was obtained from the American tissue Culture Collection.
  • Gynecological cancer (cervical, ovarian, vulvar, and endometrial) was obtained during normal surgical procedures (University of Kansas Medical Center).
  • MAb 5023 and MAb 5025 are from the DuPont Company. (See AIDS Res and Human Retroviruses 1990;6:1115-1123.) MAb 0.5b against the region was obtained from Dr. S. Matsushita of the Kumamoto University Medical School in Japan. (See AIDS Res and Human Retroviruses 1988;4:187-197.) VM77 (307- 328) was obtained from Dr. F. Veronese of Bionetics Research, Inc. Human antibodies to e ⁇ v-2-3 were provided by Dr. K.S. Steimer, Chiron Research Laboratories, Emeryville, CA.
  • Antibodies to env-2-3 represent a fraction of pooled sera from humans identified and confirmed to be seropositive in HIV-I serological assays, obtained by purification on the affinity column containing an unglycosylated form of HIV-I envelope protein gpl20 which was produced in genetically engineered yeast.
  • Iodination of MAb was routinely performed by the IODOGEN method.
  • Specific activity of MAb was 10-20 cpm/pg, and of human antibodies, 3-6 cpm/pg IgG.
  • Cells grown as confluent monolayers were seeded in a fresh medium at a density of 10 5 cells/per cm 2 in a Nunclone (Denmark) flask. After 24 h, 125 I- labeled mouse MAb or 125 I-labeled human IgG was added at a concentration of 100-300 ng/ml and cells (10-20 x 10 6 ) were labeled for 24 h. Cell fractions were obtained as described in Arch Biochem Biophys , 1989 ; 271:366-379.
  • the nuclear pellet was first washed with 0.2 M sucrose/3 mM CaCl 2 /50 mM Tris-HCl (pH 7.6) and then with 0.14 M NaCl/10 mM Tris-HCl (pH 8.3) and centrifuged at 700 g for 10 min.
  • Nucleoplasmic proteins extracted with 0.14 M NaCl were defined as the "sap protein" fraction, the pellet was swollen in a small amount of 1 mM Tris- HCl (pH 7.9) and centrifuged throughout the 1.7 M sucrose, 10 mM Tris-HCl (pH 7), at 160,000 g for 80 min. Chromatin was pelleted at the bottom of the tube, and nuclear membranes were taken at the interface.
  • Nucleoplasm (the residual fraction after extraction with 0.14 M NaCl ) was recovered from the top, and mixed with the "sap protein" fraction. Radioactivity of 125 I-MAb bound to the particular cell fractions was calculated using Avogadro's number and specific activity of the 125 I-MAb.
  • Intact nuclei were isolated by homogenization in 0.25 M sucrose, 10 mM KC1, 1.5 mM MgCl 2 , 10 mM Tris-HCl (pH 7.6), 12 mM 2-mercaptoethanol, 0.02% Triton X-100, centrifugation at 600 g for 10 min, and purification through 2.2 M sucrose, 10 mM Tris- HCl (pH 7.9), 1.5 mM MgCl 2 (90,000 g for 60 min).
  • Nuclei (2-3 x 10 6 ) were incubated with 125 I-MAb (10 ng/ml) in an incubation medium containing 0.25 M sucrose, 20 mM Tris-HCl (pH7.8), 10 mM MgCl 2 , and 500 ng/ml unlabeled bovine serum albumin.
  • nuclei were centrifuged (600 g for 10 min), washed 3 times with 50 mM Tris-HCl (pH 7.5), 12.5 mM NaCl, 12.5 mM MgCl2, homogenized in 1 mM Tris-HCl (pH 7.6), and centrifuged through 1.7 M sucrose and 10 mM Tris-HCl (pH 7.9). Nucleoplasm was taken from the top, nuclear membranes from the interface, and chromatin from the bottom of the tube. Radioactivity of 125 I-MAb in the indicated nuclear fractions was measured and the number of molecules was calculated as described (Narod SA, et al. Lancet, 338, 82-83, 1991).
  • WGA wheat germ agglutinin
  • Blotting of proteins from the polyacrylamide gel to the nitrocellulose or a PVDF membrane was performed in the TBST buffer containing 10 mM Tris- HCl (pH 8), 150 mM NaCl, and 0.05% Tween 20.
  • SKBr5 cells were incubated for 18 h with [ 35 S]methionine (10 mCi/ml, specific activity 1000 Ci/mmol) and fractionated into cytoplasm, nucleoplasm, nuclear membranes, and the chromatin described above. Cytoplasm was centrifuged (105,000 g, 1 h) , and a pellet containing microsomal fraction was dissolved in 10 mM Tris (pH 7.4), 0.5% Nonidet NP40, 0.14 M NaCl, 5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride.
  • the solubilized microsomal fraction containing cytoplasmic membranes was incubated with MAb 5023 against the HIV-I gpl20 (2-5 mg/membranes from 5 x 10 5 cells) for 1 h at 4*C with formalin-fixed Staphyloccus aureus (Calbiochem) . After incubation, S. aureus containing the MAb-cell surface protein complexes was washed with 10 mM Tris-HCl (pH 7.4), 0.5% Nonidet NP40, 0.1% SDS, and 0.14 M NaCl. Immunoprecipated proteins were analyzed electrophoretically according to Laemmli (Nature 1971;227:680-685).
  • SKBr5 cells were incubated 1 hr or 24 hr in the cell culture media containing [5,6 3H]uridine (Amersham,sp act 48 Ci/mmol) and 0 or 100 ng/ml of MAb 5023. After the incubation, cells were fractionated into the cytoplasm, nucleoplasm, nuclear membranes and chromatin as described above. Radioactivity of chromatin-bound RNA, nucleoplasmic RNA, and cytoplasmic RNA was tested in the fraction precipitated with 10% trichloroacetic acid and filtered on Whatman GF/C filters.
  • MAb 5023 and four other MAb were tested in Western blotting for reactivity with plasma membrane proteins and chromatin proteins of breast carcinoma SKBr5 (Fig. 3).
  • High molecular weight proteins (200,000 M r and 45,000 M r ) were separated by electrophoresis in 7.5% polyacrylamide gel (Fig. 3A) , and low molecular weight proteins by electrophoresis in 13% polyacrylamide gel (Fig. 3B).
  • the MAb 5023 When the electrophoresis of proteins was performed in 7.5% polyacrylamide gel, the MAb 5023 reacted with a major 160,000 M r (pl60) antigen of plasma membranes, a minor band of the M r 80,000 (p80), and a sharp band of the M r , 45,000 (p45) (Fig. 3A, lane 2).
  • Other MAbs against gpl20 recognized p45, but not pl60 and p80 (Fig. 3A, lanes 3-5). None of the protein bands detected in a plasma membrane fraction was detected in the chromatin (Fig. 3A, lane 1).
  • the typical profile of the Western reaction of MAb 5023 with the membrane protein was the same in the presence of the 1% 2-mercaptoethanol as in its absence. However, when the concentration of the 2- mercaptoethanol increased to 5%, p80 was present in higher concentrations than pl60 (not shown).
  • Immunoprecipitation of the plasma membrane fraction from [ 35 S]methionine-labeled SKBr5 cells with MAb 5023 revealed pl60, p45 (Fig. 5, lane 1), and p24 (not shown) . No proteins were immunoprecipitated by MAb 5023 from colorectal carcinoma SW707 cells (Fig. 5, lane 2), which confirms specificity of MAb 5023 reactivity with breast carcinoma antigens.
  • the protein p80 detected by Western blotting in the plasma membrane fraction of breast carcinoma cells was not detected in the immunoprecipitate.
  • pl60 represents a dimeric form of p80, where the monomer p80 in the native form is not recognized by MAb 5023.
  • MAb 5023 We also cannot eliminate the possibility that p45 and p24 in the cell membrane represent degradation products of pl60.
  • all of the proteins recognized by MAb 5023 may originate from a one precursor protein.
  • the relative amount of pl60, p80, and p24 was similar in samples obtained from independent experiments. The relative amount of p45 varied from experiment to experiment.
  • HIV-I neutralizing human antibodies to e ⁇ v-2-3 ( see Methods ) reacted in Western blotting with 80,000 M r and 45,000 M r breast carcinoma cell membrane antigens (Fig. 6). it was likely, therefore, that a fraction of human antibodies to HIV-I recognized an epitope on breast carcinoma cells also recognized by MAb 5023.
  • antibodies to env-2-3 were labeled with 125 I and incubated with SKBrS cells. A fraction of antibodies was found in the cytoplasm and in the nucleus (Table 1). The studies indicate that a fraction of human anti-HIV-I antibodies is able to penetrate breast carcinoma cells.
  • Gynecological cancer was obtained during standard surgical procedures performed in the Clinic of the Department of Obstetrics and Gynecology, University of Kansas Medical Center.
  • MAb 5023 detected antigen pl20 in 5 of 6 tested ovarian cancer tissues, in 4 of 6 cervical cancer, endometrial cancer, vulvar cancer, and pelvic cancer of an unknown origin (Fig. 7). No proteins were specifically detected in rectal cancer.
  • protein p41 was detected in several cancers. Most of gynecological cancer tissues express also p24 in both cytoplasmic and nuclear fractions, while other proteins were detected only in the cytoplasm (Fig. 7 A, B, C).
  • Other MAb against HIV-I like MAb 5025 did not detect any proteins.
  • RNA synthesis measured as [3H]uridine incorporation into TCA-precipitable fraction, increased by 25% after lhr, and by 40% after 24hr of cell exposure, compared to cells not exposed to MAb 5023 (Table 2) .
  • MAb 5023 when added to the cell culture medium at the concentration lOOng/ml, stimulated cell proliferation by 50% (Table 3). In contrast to the growth promoting action of MAb 5023 in breast cancer cells, growth of cervical cancer cell line SiHa which expresses a moderate level of pl20 was by 30%-
  • MAb developed against HIV-I gpl20 were found to cross-react with antigens of breast carcinoma cell lines and with antigens expressed in gynecological cancer.
  • MAb 5023 developed against amino acid residues 308-322 of the variable V3 loop of HIV-I reacted in Western blotting with a 160,000 M r (P160) AND 80,000 M--(p80) cell surface antigens. Protein pl60 seems to represent an oligomeric form of p80.
  • MAb 5023 detects a 120,00 M-. (pl20) and a 41,000 M r (p41) proteins.
  • MAb 5025 directed against the same region 308-322, and MAb against amino acid regions 307-328 and 308-332, did not recognize pl60 or p80. It was shown before that although MAb 5023 and MAb 5025 were developed against the same synthetic peptide, there are minor differrrences in the structure of the core epitope recognized by these MAb, which account for the several-fold higher affinity of MAb 5023 than that of MAb 5025. All of the MAb we tested recognized another cell membrane antigen, p45 in breast cancer and p41 in gynecological cancer. It is not established whether p45 and p41 represent independent proteins, a degradation, or processing products of pl60 and pl20 respectively.
  • MAb 5023 was the only MAb which was able to enter breast carcinoma cells and translocate to the nucleus, it is likely that pl60 (and p80) expresses a specific epitope which is critical for MAb internalization.
  • An unspecific adsorption of the cell membrane-bound 125 I-MAb 5023 to the chromatin during cell fractionation may be eliminated, since pl60, p80, and p45 and pl20 and p41 represent the specific markers of the cell membrane fraction and are not found in the chromatin.
  • the fact that MAb 5023, but none of the other MAb, was internalized suggests that an antibody binding to a cell surface antigen is not sufficient to induce the process of internalization. Instead, a specific epitope of the cell surface antigen must be involved. The latest observation is consistent with our previous studies, which proved that only a few from many MAb developed against tumor-a ⁇ sociated antigens are internalized, while others are unable to enter the cell.
  • MAb 5023 but none of the other MAb, recognized p24 in the cell membrane fraction and in the chromatin.
  • chromatin also expresses a minor p23 band.
  • the chromatin did not show any trace of pl60, p80, and p45, which eliminates the possibility that the cytoplasmic protein p24 attached unspecifically to the chromatin during cell fractionation.
  • p24 may be involved in binding the translocated MAb 5023 to the chromatin. It seems that p24 represents an antigen expressed on the cell surface, as well as in the chromatin of breast carcinoma cells and gynecological cancer cells.
  • a weak expression of p24 was also observed in the chromatin of 451 in melanoma cells (Fig. 4B; lane 5). Chromatin of T lymphocytes did not express p24/p23. It is noteworthy that low expression of p24 was detectable in the plasma membrane fraction of several T lymphocyte cell lines. Binding of MAb 5023 to p24 was specifically inhibited by the synthetic peptide RIQRGPGRAFVTIGK, towards which the MAb 5023 was developed.
  • the internalized MAb 5023 was developed against a short region of HIV-I gpl20, covering amino acids 308-322 (RIQRGPGRAFVTIGK), but this MAb binds to the much shorter amino acid region GRAF. It is likely that this core epitope must also be expressed in breast carcinoma pl60. Alternatively, gpl60 may express a conformation epitope homologous to that of HIV-I gpl20. Whether the proteins of similar M-. in breast carcinoma and HIV-I that cross-react with MAb against HIV-I gpl20 represent products of human or retrovirus genes is currently unknown. We suspect that a retrovirus of strong homology to HIV-I may be present in breast carcinomas.
  • Antigens pl60 and pl20 were selectively detected in the cytoplasmic fraction, while proteins p42 and p25 were detected in both cytoplasmic and chromatin fractions (Fig. 9 & 10).
  • Normal ovarian tissue Fig. 9B
  • skin, muscles, normal cervical tissue and normal vaginal mucosa tested negative Fig. 9C
  • MAb anti-HIV-I reacted selectively with cancer.
  • Melanoma, lung carcinoma and colorectal carcinoma also did not react with MAb anti-HIV-I gpl20 (Rakowicz-Szulczynska EM, et al., in Nuclear Localization of Growth Factors and of Monoclonal Antibodies . ed. E.M.
  • MAb which recognized pl60, pl20, p42 and p24 in cancer cells, was developed against amino acid sequences 308-322 (RIQROPQRATVTIGK) SEQ ID NO:l of the variable loop of HIV-I gpl20, and this MAb binds to the epitope GRAF (Durda PJ, Bacheler L, Clapham P, Jenow ⁇ ki AM, Leece B, Matthews TJ, McKnoght A, Pomerantz R, Rayner M & Weinhold KJ. AIDS Res Human Retroviruses, 6, 1115-1118, 1988; Langedijk JPM, Back NKT, Durda PJ, Goudsmit J & Meloen RH. J Gen Virol .
  • RIQROPQRATVTIGK amino acid sequences 308-322
  • Cytoplasm isolated from mixed Mullerian tumor, was electrophoretically separated in 10% polyacrylamide gel with SDS, blotted into the PVDF membrane and exposed to anti-HIV-I gpl20 MAb which was preincubated (Fig. 10B) or not preincubated (Fig. 10A) with the peptide.
  • Antigens pl20, p42 and p25 strongly reacted with MAb anti- HIV-I gpl20 which was not preincubated (Fig. 10A) , but did not react with MAb which was preincubated with the peptide (Fig. 10B).
  • the peptide RIQRGPGRAFVTIGK SEQ ID NO: 1 competitively blocked binding of the MAb to the cancer antigens (Fig.
  • PCR polymerase chain reaction
  • SEQ ID NO: 3 consisted of two 20-mers derived from the gene for envelope protein gp41 of HIV-I (Laure F, Courgnaud V, Rouzioux C, Blanche S, Veber F, Burgard M, Jacomet C, Griscelli C & Brechot C. Lancet, 2, 538, 1988).
  • the second set of primers (P1/P2): PI (764-786, region Gag) :
  • SEQ ID NO:5 was derived from the Gag gene of HIV-I (Ou CY, Kwok S, Mitchell SW, Mack DH, Sninsky JJ, Krebs JW, Feorino P, Warfield D & Schochetman G. Science. 239, 295, 1988). Both sets of HIV-derived primers initiated the PCR in the presence of DNA isolated either from HIV-I infected lymphocytes, or from breast cancer MCF7, cervical cancer SiHa cells, endometrial cancer or mixed Mullerian tumor obtained during surgery (Fig. 11). Negative reaction was obtained with control DNA isolated from normal skin (Fig. 11). PCR with DNA isolated from noninfected lymphocytes is reportedly negative (Laure F, et al.. Lancet. 2, 538, 1988) and was also negative in our studies (not shown).
  • the PCR products obtained with primers SK 68/SK 69 were of the same size (approximately 140bp) in the case of MCF7, SiHa and gynecological cancer DNA, as in the case of the DNA isolated from HIV-infected cells which was used as the template (Fig. 11A) .
  • the sequential analysis of the amplified fragments revealed 140bp of cancer DNA with no significant homology to any known human gene. Of the 140bp, 105bp were identical in at least two of three cancers tested and 53bp were identical in all three cancers (Fig. 12). 21bp located at the 3' end of the amplified region of MCG7 breast cancer DNA were identical to the HIV sequences (Fig. 12).
  • SiHa cell DNA 20 of 21bp were identical to HIV sequences.
  • 19bp were identical to HIV-I sequences, 2bp were different and 2bp were inserted within the region of homology with HIV-I, MCF7 and SiHa.
  • the DNA fragments amplified by PCR in the presence of the second set of primers (Pl/P2-Gag derived) was only 130bp-long in breast, cervical and endometrial cancer DNA, comparing to the 304bp-long fragment obtained with DNA isolated from HIV-I infected cells (Fig. 1 IB) and showed a limited homology with HIV-I genome (not shown).
  • SK68/SK69 primers derived from HIV-I successfully amplified DNA sequences from breast cancer tissues taken from seven patients ( Figure 15).
  • SK68/SK69 primers also amplified DNA sequences from prostate cancer tissues taken from six patients ( Figure 16).
  • Visualization of a cross- section Fig.
  • FIG. 12D of the large cytoplasmic vesicle, located close to the cell surface, revealed several oval-shaped, viral-like particles localized within the vesicle.
  • the viral-like particles were also "budding" from the extracellular vacuoles (Fig. 12B) .
  • the size of the particles was approximately 120 - 150nm.
  • Immuno-gold labeling of the isolated viral particles with MAb anti-gpl20 strongly supports the hypothesis about the viral origin of HIV-crossreactive cancer antigens and is consistent with genetic homology observed on the DNA level. Analysis of the localization of the virus- like particles strongly suggests that the virus is either synthesized inside the membrane-coated vesicles or, after synthesis, buds into the intracytoplasmic vacuoles , which are then secreted to the cell surface, fuse with the membrane and release viral particles through exocytosis. Exocytosis of the virus-containing vesicles might be responsible for the formation of the characteristic "peninsula-like" surface of the cancer cells, with very long villi-like structures.
  • D retrovirus is associated with Simian AIDS and was first isolated in 1970 from a naturally occurring breast carcinoma in a captive rhesus macaque (Chopra HC & Mason MM. Cancer Res . 30, 2081-2086, 1970).
  • Several laboratories demonstrated D-type virus in established human cell lines Ilyin KV, Bykovsky AF S. Zhdanov VM. CA, 32, 89-96, 1973; Porovic M, Kalyanaraman VS, Reitz MS & Sarngadharan MG. Am J Cancer, 30, 93-99, 1982), however, origin of these viruses was never established.
  • Rakowicz-Szulczynska CRC Press, pp.180-197, 1993; Rakowicz-Szulczynska E, Raso V, Kaczmarski W & Durda PJ. Antibody Immunocon Radiopharm, 6, 209- 219, 1993; Rakowicz-Szulczynska EM, Mclntosh DG & Smith ML. Infect Pis Obstet Gvnecol. 1994, in press; Rakowicz-Szulczynska EM, Mclntosh DG & Smith ML. Mechanisms of cancer growth promotion by HIV-I neutralizing antibodies, submitted to Breast Cancer Res Treatment, 1994).
  • antisense oligonucleotides complementary to the identified sequences of cancer DNA, have been synthesized and used to treat SiHa and MCF7 cells in vitro (Fig. 13).
  • Antisense oligonucleotides RAK-I (21-mer 5'- CCAGACTGTGAGTTGCAACAG-3 ' ) SEQ ID NO: 6, RAK-II (15- mer 5 '-CAACAGCCTACAACC-3 ' ) SEQ ID NO: 7 and RAK-III (15-mer 5 * -TCTTCTAATCCCAAA-3 ' ) SEQ ID NO: 8, complementary to the cancer DNA sequences amplified with HIV-I derived primers, inhibited growth of cells by 50, 70 and 30%, respectively, within 4 days (Fig. 13). Control antisense oligonucleotides (5'- TGTGACATCAGGCTCAAATC-3' ) did not inhibit cell growth.
  • the cancer virus seems to contain a hybrid genome with homology to different isolates of HIV, since the MAb anti-HIV-I gpl20 was developed against the gpl20 variable domain of HIV-I IIIb , and PCR primers SK 68/SK 69 originated from the isolate HIV- I ARV - 2 -
  • the 21bp segment of breast, cervical and endometrial cancer DNA, identical to HIV sequences, is highly conserved in different strains of HIV-I. The sequences preceding the conserved segment were, to a certain degree, homologous to several HIV-I strains.
  • the HIV-like antigens pl20, p42 and p25 were detected selectively in breast and gynecological cancer, but not in normal tissues and, therefore, represent unique markers of female malignancies.
  • One of the HIV-crossreactive antigens is detectable in the blood of breast cancer patients and it seems to represent the novel marker of spontaneous female malignancies. Since antisense oligonucleotide RAK- I, II and III inhibited growth of breast and cervical cancer cells in vitro, the novel Female Cancer Virus represent a promising target for cancer diagnosis, prognosis and therapy.
  • MOLECULE TYPE protein
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • FRAGMENT TYPE internal
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE NO
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • SEQUENCE DESCRIPTION SEQ ID NO: 6: CCAGACTGTG AGTTGCAACA G 21
  • MOLECULE TYPE DNA (genomic.)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES
  • MOLECULE TYPE DNA (genomic)
  • HYPOTHETICAL NO
  • ANTI-SENSE YES

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Abstract

L'invention concerne de nouvelles séquences d'ADN et des poylpeptides qui sont associés aux cancers du sein, aux cancers gynécologiques et au cancer de la prostate. L'invention concerne également de techniques permettant de détecter et de traiter les cancers du sein, les cancers gynécologiques et les cancers de la prostate. Dans un mode de réalisation préféré, ces techniques comprennent l'utilisation d'un anticorps monoclonal développé contre un peptide synthétique correspondant à une protéine du virus de l'immunodéficience humaine (VIH-1). Dans un mode de réalisation encore préféré au précédent, l'anticorps est dirigé contre la protéine enveloppe gp120 (région d'acides aminés 308-322). Les régions d'homologie entre le VIH-1 et les séquences du cancer du sein et des cancers gynécologiques sont identifiés ainsi, que des méthodes de diagnostic et de traitement basées sur les régions conservées.
PCT/US1998/015580 1997-07-29 1998-07-29 Detection et traitement du cancer du sein, des cancers gynecologiques et du cancer de la prostate WO1999009047A1 (fr)

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EP98938042A EP1012159A1 (fr) 1997-07-29 1998-07-29 Detection et traitement du cancer du sein, des cancers gynecologiques et du cancer de la prostate
CA002297727A CA2297727A1 (fr) 1997-07-29 1998-07-29 Detection et traitement du cancer du sein, des cancers gynecologiques et du cancer de la prostate
AU86654/98A AU8665498A (en) 1997-07-29 1998-07-29 Detection and treatment of breast, gynecological and prostate cancer

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995010777A1 (fr) * 1993-10-15 1995-04-20 Rakowicz Szulczynska Ewa M Detection et traitement des cancers du sein et des cancers gynecologiques

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995010777A1 (fr) * 1993-10-15 1995-04-20 Rakowicz Szulczynska Ewa M Detection et traitement des cancers du sein et des cancers gynecologiques

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
RAKOWICZ-SZULCZYNSKA E. M., ET AL.: "ANTIGEN RAK: A NEW BREAST CANCER DIAGNOSTIC MARKER.", JOURNAL OF TUMOR MARKER ONCOLOGY., LIEBERT, NEW YORK, NY., US, vol. 10., no. 03., 1 January 1995 (1995-01-01), US, pages 25 - 37., XP002915153, ISSN: 0886-3849 *
RAKOWICZ-SZULCZYNSKA E. M., ET AL.: "DIAGNOSTIC EVALUATION OF CANCER ANTIGENS RAK I. CERVICAL AND OVARIAN CANCER.", INTERNATIONAL JOURNAL OF ONCOLOGY, DEMETRIOS A. SPANDIDOS ED. & PUB, GR, vol. 09., no. 04., 1 October 1996 (1996-10-01), GR, pages 693 - 699., XP002915152, ISSN: 1019-6439 *

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