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WO2003034068A1 - Localisation de troubles associes aux kallikreines - Google Patents

Localisation de troubles associes aux kallikreines Download PDF

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Publication number
WO2003034068A1
WO2003034068A1 PCT/CA2002/001556 CA0201556W WO03034068A1 WO 2003034068 A1 WO2003034068 A1 WO 2003034068A1 CA 0201556 W CA0201556 W CA 0201556W WO 03034068 A1 WO03034068 A1 WO 03034068A1
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WIPO (PCT)
Prior art keywords
kallikrein
imaging
tumor
image
agent
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PCT/CA2002/001556
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English (en)
Inventor
Eleftherios P. Diamandis
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Mount Sinai Hospital
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Priority claimed from PCT/CA2001/001505 external-priority patent/WO2002035232A2/fr
Application filed by Mount Sinai Hospital filed Critical Mount Sinai Hospital
Publication of WO2003034068A1 publication Critical patent/WO2003034068A1/fr

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B82NANOTECHNOLOGY
    • B82YSPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6891Pre-targeting systems involving an antibody for targeting specific cells
    • A61K47/6897Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies
    • A61K47/6898Pre-targeting systems with two or three steps using antibody conjugates; Ligand-antiligand therapies using avidin- or biotin-conjugated antibodies
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00Preparations for testing in vivo
    • A61K49/06Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/08Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by the carrier
    • A61K49/10Organic compounds
    • A61K49/14Peptides, e.g. proteins
    • A61K49/16Antibodies; Immunoglobulins; Fragments thereof
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02Preparations 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
    • A61K51/04Organic compounds
    • A61K51/08Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10Antibodies 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
    • A61K51/1045Antibodies 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
    • A61K51/1072Antibodies 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 the tumor cell being from the reproductive system, e.g. ovaria, uterus, testes or prostate
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407Specifically defined cancers
    • G01N33/57449Specifically defined cancers of ovaries
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/914Hydrolases (3)
    • G01N2333/948Hydrolases (3) acting on peptide bonds (3.4)
    • G01N2333/95Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433Serine endopeptidases (3.4.21)
    • G01N2333/96441Serine endopeptidases (3.4.21) with definite EC number
    • G01N2333/96455Kallikrein (3.4.21.34; 3.4.21.35)

Definitions

  • TITLE Localization of Disorders Associated with Kallikreins FIELD OF THE INVENTION
  • the invention relates to the localization of disorders associated with kallikreins, in particular kalllikrein
  • pancreatic/renal kallikrein (KLK1, encoding for hKl protein), human glandular kallikrein 2 (KLK2, encoding for hK2 protein) and human kallikrein 3 (KLK3, encoding for hK3 protein or prostate-specific antigen, PSA).
  • KLK1, encoding for hKl protein pancreatic/renal kallikrein
  • KLK2, encoding for hK2 protein human glandular kallikrein 2
  • KLK3, encoding for hK3 protein or prostate-specific antigen, PSA prostate-specific antigen
  • the KLK6 gene (encoding for human kallikrein 6, hK6) has been cloned independently by three groups of investigators and was previously given the names zyme (Little et al. 1997) [cloned from brain tissue], protease M (Anisowicz et al. 1996) [cloned from breast tissue] and neurosin (Yamashiro et al. 1997) [cloned from a colon carcinoma cell line]. Recently, uniform nomenclature for all newly discovered and the traditional kallikrein genes has been established (Diamandis et al. 2000b).
  • the KLK6 gene encodes for a trypsin-like serine protease of 244 amino acids in length, of which 16 amino acids constitute the signal peptide and 5 amino acids, the activation peptide.
  • the mature enzyme consists of 223 amino acids. It has been previously predicted that hK6 is a secreted protein (Little et al. 1997; Anisowicz et al. 1996; Yamashiro et al. 1997; Yousef et al. 1999).
  • the genomic organization and the hormonal regulation of the KLK6 gene has been investigated and its tissue expression by reverse-transcription-polymerase chain-reaction has been studied (Yousef et al. 1999).
  • the gene is abundantly expressed in many tissues, including the salivary gland, brain, uterus, heart, thymus, prostate, liver, breast, thyroid, spleen, placenta, trachea, testis and kidney (Yousef et al. 1999).
  • the invention relates to a method for detecting and locating disorders associated with one or more, preferably two or more, kallikreins.
  • the invention contemplates compositions and methods for imaging tumor cells and tissues in vivo and in situ, which can be particularly useful for imaging abnormal tissue and organs, including sites of primary and metastatic tumors.
  • the disorder is associated with kallikrein 6 (hK6) and may additionally include one or more of kallikrein 4 (hK4), kallikrein 5 (hK5), kallikrein 7 (hK7), kallikrein 8 (hK8), kallikrein 9 (hK9), kallikrein 10 (hK10), kallikrein 11 (hKl l), kallikrein 12 (hK12), kallikrein 13 (hK13), kallikrein 14 (hK14), and kallikrein 15 (hK15).
  • the disorder is cancer, more preferably ovarian cancer, most preferably epithelial ovarian cancer.
  • the invention includes a method of identifying the presence of a tumor tissue bearing a kallikrein in an excised tissue sample from a subject (e.g. biopsy sample) comprising the steps of: exposing the tissue to an amount of a detectably-labeled kallikrein binding molecule or agent effective to bind to the kallikrein; and examining the sample for the presence or absence of bound labeled kallikrein.
  • the invention also provides a method of evaluating an excised mammalian tissue sample for the presence of a tumor bearing a kallikrein (e.g. ovarian tumor) comprising the steps of: exposing the tissue to an amount of a detectably labeled kallikrein binding molecule or agent effective to bind to the kallikrein tumor tissue, and examining the sample for the presence or absence of labeled kallikrein.
  • a kallikrein e.g. ovarian tumor
  • an in vivo method comprising administering to a subject an agent that has been constructed to target one or more kallikreins.
  • the invention contemplates an in vivo method comprising administering to a mammal one or more agent that carries a label for imaging and binds to a kallikrein, preferably hK6, and then imaging the mammal.
  • the invention provides a method of imaging a tumor, preferably an ovarian tumor in a subject, the tumor having one or more kallikreins selected from the group consisting of hK4, hK5, hK6, hK7, hK8, hK9, hK10, hKl l, hK12, hK13, hK14, and hK15, comprising the steps of: delivering into the subject an amount of a detectably-labeled kallikrein binding molecule effective to image the tumor; and scanning the subject to determine the distribution of the labeled kallikrein.
  • the tumor is an ovarian tumor having hK5 and hK6; hK4, hK5, hK6, hK7, and hK8;.or, hK4, hK5, hK6, hK7, hK8, hK9, hK10, and hKl 1.
  • an in vivo method for imaging ovarian cancer comprising: (a) injecting a patient with an agent that binds to kallikrein 6 the agent carrying a label for imaging the ovarian cancer;
  • a method for imaging ovarian cancer may further comprise in step (a) above, injecting the patient with one or more of an agent that binds to human stratum corneum chymotryptic enzyme (HSCCE, also known as kallikrein 7 or hK7), kallikrein 4, kallikrein 5, kallikrein 8, kallikrein 9, kallikrein 10, kallikrein 11, kallikrein 12, kallikrein 13, kallikrein 14, kallikrein 15, CA125, CA15-3, CA19-9, CA72-4, OVX1, lysophosphatidic acid (LPA) or carcinoembryonic antigen (CEA).
  • HSCCE human stratum corneum chymotryptic enzyme
  • composition adapted for imaging ovarian cancer comprising an agent that binds to one or more of hK4, hK5, hK6, hK7, hK8, hK9, hK10, hKll, hK12, hK13, hK14, and hK15, and a label for imaging is provided to a patient or to a tissue sample, and the kallikrein is located in the patient or sample by visualizing the imaging agent bound to the kallikrein.
  • the composition comprises an agent that binds to hK6 and a label for imaging, and an agent that binds to one or more of hK4, hK5, hK7, hK8, hK9, hK10, and hKll, wherein the agent for hK4, hK5, hK7, hK8, hK9, hK10, and hKll is labeled so that each agent is distinguished during the imaging.
  • the composition comprises agents that bind to hK5 and hK6, or hK4, hK5, hK6, hK7, and hK8.
  • the invention also provides a nucleic acid encoding a chi eric polypeptide comprising a substance that binds to a kallikrein (e.g. hK6) and a label for imaging the kallikrein.
  • the invention also provides an expression vector comprising a nucleic acid encoding such a chimeric polypeptide.
  • the invention provides a cell e.g. a transformed viral-infected cell, comprising a nucleic acid encoding a chimeric polypeptide of the invention.
  • the cell can be a bacterial, a yeast, an insect, or a mammalian cell.
  • the invention provides a recombinant chimeric polypeptide produced by such a cell.
  • the invention still further provides a pharmaceutical formulation comprising a composition comprising an agent that binds a kallikrein (e.g. anti-hK6) and a label for imaging a tumor, preferably an ovarian tumor, and a pharmaceutically acceptable excipient, and wherein the composition is present in an amount sufficient to enhance a computer assisted tomography (CAT) image, a magnetic resonance spectroscopy (MRS) image, a magnetic resonance imaging (MRI) image, a positron emission tomography (PET) image, a single-photon emision computed tomography (SPECT) image, or a bioluminescence image (BLI), or equivalents thereof, when the pharmaceutical formulation is administered to an individual, or applied to a tissue or organ in situ, in a sufficient amount.
  • CAT computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emision computed tomography
  • BBI
  • the invention contemplates a method for in situ or in vivo imaging of a cell, tissue, an organ, or a full body comprising administering a pharmaceutical formulation of the invention, in an amount sufficient to enhance an image, wherein the image is generated by computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS) image, magnetic resonance imaging (MRI), positron emission tomography (PET), single- photon emision computed tomography (SPECT), or bioluminescence imaging (BLI), or equivalents thereof.
  • CAT computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single- photon emision computed tomography
  • BBI bioluminescence imaging
  • the invention also provides a method of in situ or in vivo imaging of a cell, a tissue, an organ or a full body comprising the following steps: (a) providing a pharmaceutical formulation of the invention; (b) providing an imaging device, wherein the imaging device is computer assisted tomography (PET), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), a positron emission tomography (PET), single-photon emision computed tomography (SPECT), or bioluminescence imaging (BLI) or equivalent; (c) administering the pharmaceutical formulation in an amount sufficient to generate the cell, tissue, or body image; and (d) imaging the distribution of the pharmaceutical formulation of step (a) with the imaging device, thereby imaging the cell, tissue or body.
  • PET computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emision computed tomography
  • BLI bioluminescence imaging
  • the pharmaceutical formulation is administered to a human, such as a cancer patient, in particular an ovarian cancer patient, or a patient suspected of having or being screened for cancer, in particular ovarian cancer.
  • a human such as a cancer patient, in particular an ovarian cancer patient, or a patient suspected of having or being screened for cancer, in particular ovarian cancer.
  • the pharmaceutical formulation is administered intravenously.
  • the image may be taken between 2 minutes and 24 hours after administration of the pharmaceutical formulation.
  • the invention contemplates a method for in vivo imaging tumor neovasculature in a subject comprising: (a) providing a pharmaceutical formulation of the invention; (b) providing an imaging device, wherein the imaging device is computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), bioluminescence imaging (BLI) or equivalent; (c) administering the pharmaceutical formulation in an amount sufficient to image the tumor neovasculature; and, (d) imaging the distribution of the pharmaceutical formulation of step (a) with the imaging device, thereby imaging the tumor neovasculature.
  • CAT computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • BLI bioluminescence imaging
  • the invention provides a method for in situ or in vivo screening for an anti-tumor agent by imaging tumor neovasculature in an individual comprising the following steps: (a) providing a composition comprising a chimeric polypeptide or a pharmaceutical formulation of the invention, and a test compound; (b) providing an imaging device, wherein the imaging device is computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT), bioluminescence imaging (BLI) or equivalent; (c) administering the composition of step (a) in an amount sufficient to image the tumor and imaging the distribution of the composition with the imaging device, thereby imaging the tumor; (d) administering the test compound; and, (e) imaging the distribution of the composition with the imaging device, thereby imaging the tumor, wherein a decrease in the amount of tumor neovasculature indicates that the compound is an anti-tumor or an anti-angi
  • the agent or kallikrein binding molecule is an antibody that recognizes or binds the kallikrein.
  • the agent is a chemical entity which recognizes or binds the kallikrein.
  • the agent or kallikrein binding molecule carries a label to image the kallikreins.
  • labels useful for imaging in accordance with the present invention are radiolabels, fluorescent labels (e.g fluorescein and rhodamine), nuclear magnetic resonance active labels, positron emitting isotopes detectable by a positron emission tomography (“PET”) scanner, chemiluminescers such as luciferin, bioluminescent labels, and enzymatic markers such as peroxidase or phosphatase.
  • PET positron emission tomography
  • Short-range radiation emitters such as isotopes detectable by short-range detector probes can also be employed.
  • kits for carrying out the methods of the invention contemplates kits comprising the pharmaceutical formulations, chimeric polypeptides, or nucleic acids encoding the chimeric polypeptides of the invention.
  • Figure 1(A) is a graph showing the frequency distribution of hK6 specific activity in ovarian tumor extracts.
  • the value of 35ng/mg of total protein corresponds to the limit that, according to Chi square analysis, gives the best prediction of overall survival of the study population. See Figure 1(B) for Chi square plot.
  • Tumors with hK6 in excess of 35 ng/mg total protein were classified as hK6 positive and those with values less than or equal to 35 ng/mg total protein were classified as hK6 negative. 30% of the tumors were classified as positive by this criterion.
  • Figure 2 is a graph showing a comparison of hK6 concentration in extracts from normal ovarian tissues ("normal"), and ovarian cancer ("cancer"). N indicates the number of specimens in each group. Horizontal bars represent the median hK6 specific activity (ng hK6/mg total extract protein) in each group. The Krustal Wallis test showed that extracted hK6 specific activity was significantly elevated in the ovarian tumor preparations (P ⁇ 0.001).
  • Figure 3 is a graph showing the distribution of hK6 specific activity (ng hK6/mg total protein) in tumor extracts from stage I/II and stage m/IV ovarian cancer patients. N indicates the number of tumors comprising each group. Horizontal bars represent the median value of hK6 tumor specific activity.
  • FIG. 4 shows Kaplan-Meier survival curves of the entire patient population under study :effect of hK6 status. Top: progression-free survival (PFS). Bottom: overall survival (OS). The patient number in each group (n) is indicated as is the statistical significance (P value) of the survival difference between hK6 positive and hK6 negative groups. The adverse effect of hK6 positivity on both time to progression and overall survival was significant.
  • Figure 5 are graphs showing the effect of hK6 status (positive or negative) on progression -free survival
  • FIG. 6 is a blot showing immunohistochemical localization of hK6 in ovarian neoplasms of varying malignant potential, cell type, and origin (epithelial versus mesenchymal).
  • A Invasive papillary serous adenocarcinoma, the common malignant epithelial tumor of the ovary.
  • D Mucinous epithelial tumor of low malignant potential, an epithelial tumor of intermediate grade. Note weak, diffuse cytoplasmic staining of neoplastic epithelium and absent staining in supportive stroma (far left).
  • the invention provides a method for imaging tumors associated with one or more kallikreins, preferably kallikreins associated with ovarian cancer, most preferably hK6, and optionally hK4, hK5, hK7, hK8, hK9, hKlO, hKll, hK12, hK13, hK14, and hK15.
  • a method for imaging ovarian cancer may further comprise providing to a patient or a tissue sample one or more of an agent that binds to human stratum corneum chymotryptic enzyme (HSCCE), kallikrein 4, kallikrein 5, kallikrein 8, kallikrein 9, kallikrein 10, kallikrein 11, CA125, CA15-3, CA19-9, OVX1, lysophosphatidic acid (LPA), or carcinoembryonic antigen (CEA), preferably hK4, hK5, hK6, hK7, and hK8.
  • HSCCE human stratum corneum chymotryptic enzyme
  • kallikrein 4 kallikrein 5, kallikrein 8, kallikrein 9, kallikrein 10, kallikrein 11, CA125, CA15-3, CA19-9
  • OVX1 lysophosphatidic acid
  • CEA carcinoembryonic antigen
  • each agent is labeled so that each marker can be distinguished during the imaging.
  • hK6 or "kallikrein 6” refers to human kallikrein 6, (also known as zyme, protease M, and neurosin) a trypsin-like serine protease of 244 amino acids in length, of which 16 amino acids constitute the signal peptide and 5 amino acids, the activation peptide (7, 8, and 9).
  • the amino acid sequence for hK6 can be found at GenBank Accession Nos. AF013988, AF149289, HSU62801, D78203, and NM002774.
  • kallikrein 4" or “hK4" refers to human kallikrein proteins that have the sequences provided in GenBank and identified by the Accession Nos. in Table 6. The kallikrein proteins are also described in the publications referenced in the GenBank records.
  • the kallikein proteins referred to herein include all homologs, naturally occurring allelic variants, isoforms and precursors of the kallikrein sequences set out in the GenBank records referred to in Table 6.
  • naturally occurring allelic variants of a human kallikrein protein will share significant homology (70-90%) to the sequences shown in the GenBank Accession Nos..
  • Allelic variants may contain conservative amino acid substitutions from the kallikrein sequences or will contain a substitution of an amino acid from a corresponding position in a kallikrein homologue such as, for example, the murine kallikrien homologue.
  • tissue samples are imaged using methods of the invention.
  • Tissue samples can be excised from a subject and treated with one or more agent that binds to a kallikrein and carries a label for imaging the kallikrein.
  • a kallikrein is located in the sample by visualizing the label bound to kallikrein in the sample.
  • the tissue sample may be a tumor tissue, or a tissue suspected of comprising tumor tissue or cells, in particular ovarian tumor tissue or cells.
  • an immunohistology method is used to assay for the presence or absence of a kallikrein in a tissue sample such as an ovarian tumor tissue sample.
  • the tissue sample may be treated with antibodies specific for a kallikrein and antibodies bound to the kallikrein can be detected by treating with a biotinylated antibody against the kallikrein specific antibody followed by treatment with an enzyme tagged avidin (e.g. avidin-peroxidase) and enzyme substrate (e.g. chromogenic peroxidase substrate).
  • the invention also provides in vivo methods whereby a subject or patient is administered one or more agents that carry an imaging label, and that are capable of targeting or binding to a kallikrein. The agent is allowed to incubate in vivo and bind to the kallikrein(s) associated with a tumor, preferably ovarian tumors.
  • the presence of the label is localized to the ovarian cancer, and the localized label is detected using imaging devices known to those skilled in the art.
  • the term "subject” or “patient” refers to a warm-blooded animal such as a mammal that is afflicted, suspected of being afflicted with, or being screened for a condition associated with a kallikrein, in particular cancer, more particularly ovarian cancer.
  • "subject” refers to a human.
  • the agent may be an antibody or chemical entity that recognizes or binds to the kallikrein(s).
  • antibody includes both intact antibodies having at least two heavy (H) chains and two light (L) chains inter- connected by disulfide bonds and antigen binding fragments thereof, or equivalents thereof, either isolated from natural sources, recombinantly generated or partially or entirely synthetic.
  • antigen binding fragments include, e.g., Fab fragments, F(ab') 2 fragments, Fd fragments, dAb fragments, isolated complementarity determining regions (CDR), single chain antibodies, chimeric antibodies, humanized antibodies, human antibodies made in non-human animals (e.g., transgenic mice) or any form of antigen binding fragment.
  • Antibodies including monoclonal and polyclonal antibodies, fragments and chimeras, may be prepared using methods known to those skilled in the art.
  • antibodies used in the methods of the invention are reactive against a kallikrein if they bind with a K a of greater than or equal to 10 "7 M.
  • An agent may be a peptide that mimics the epitope for an antibody specific for a kallikrein and binds to the kallikrein.
  • the peptide may be produced on a commercial synthesizer using conventional solid phase chemistry.
  • a peptide may be prepared that includes either tyrosine lysine, or phenylalanine to which N 2 S 2 chelate is complexed (See U.S. Patent No. 4,897,255).
  • the anti-kallikrein peptide conjugate is then combined with a radiolabel (e.g. sodium 99ra Tc pertechnetate or sodium 188 Re perrhenate) and it may be used to locate a kallilkrein producing tumor.
  • the agent carries a label to image the kallikreins.
  • the agent may be labelled for use in radionuclide imaging.
  • the agent may be directly or indirectly labelled with a radioisotope. Examples of radioisotopes that may be used in the present invention are the following: 277 Ac, 2l l At, l2S Ba, l , Ba, 7 Be, 204 Bi,
  • the radioisotope is ,3I I, ,25 I, ,23 I, , n I, 99m Tc, 90 Y, 186 Re, 188 Re, 32 P, 153 Sm, 67 Ga, 20, T1 77 Br, or l8 F, and is imaged with a photoscanning device.
  • the agent may be administered to the patient, it is localized to the tumor having a kallikrein with which the agent binds, and is detected or "imaged" in vivo using known techniques such as radionuclear scanning using e.g., a gamma camera or emission tomography.
  • radionuclear scanning e.g., a gamma camera or emission tomography.
  • a positron emission transaxial tomography scanner can also be used where the radiolabel emits positrons (e.g., " C, I8 F, Is O, and ,3 N).
  • Whole body imaging techniques using radioisotope labeled agents can be used for locating both primary tumors and tumors which have metastasized.
  • Antibodies specific for kallikreins, or fragments thereof having the same epitope specificity are bound to a suitable radioisotope, or a combination thereof, and administered parenterally.
  • administration preferably is intravenous.
  • the bio-distribution of the label can be monitored by scintigraphy, and accumulations of the label are related to the presence of ovarian cancer cells.
  • Whole body imaging techniques are described in U.S. Pat. Nos. 4,036,945 and 4,311,688.
  • agents useful for diagnosis and therapeutic use which can be coupled to antibodies and antibody fragments include metallothionein and fragments (see, U.S. Pat. No. 4,732,864). These agents are useful in diagnosis staging and visualization of cancer, in particular ovarian cancer, so that surgical and/or radiation treatment protocols can be used more efficiently.
  • the agent may carry a bioluminescent or chemiluminescent label.
  • bioluminescent or chemiluminescent labels include polypeptides known to be fluorescent, bioluminescent or chemiluminescent, or, that act as enzymes on a specific substrate (reagent), or can generate a fluorescent, bioluminescent or chemiluminescent molecule.
  • bioluminescent or chemiluminescent labels include luciferases, aequorin, obelin, mnemiopsin, berovin, a phenanthridinium ester, and variations thereof and combinations thereof.
  • the pharmaceutical formulation of the invention can further comprise a substrate for the bioluminescent or chemiluminescent polypeptide.
  • the chemiluminescent polypeptide can be luciferase and the reagent luciferin.
  • the substrate can be administered before, at the same time (e.g., in the same formulation), or after administration of the chimeric polypeptide (including the enzyme).
  • a substrate for a bioluminescent or chemiluminescent label can be administered before, at the same time
  • the invention contemplates a pharmaceutical formulation comprising an agent labeled with a bioluminescent or chemiluminescent label and further comprising a substrate for the bioluminescent or chemiluminescent polypeptide.
  • An agent may comprise a paramagnetic compound, such as a polypeptide chelated to a metal, e.g., a metalloporphyrin.
  • the paramagnetic compound may also comprise a monocrystalline nanoparticle, e.g., a nanoparticle comprising a lanthanide (e.g., Gd) or iron oxide; or, a metal ion comprising a lanthanide.
  • lanthanide e.g., Gd
  • “Lanthanides” refers to elements of atomic numbers 58 to 70, a transition metal of atomic numbers 21 to 29, 42 or 44, a Gd(III), a Mn(II), or an element comprising an Fe element.
  • Paramagnetic compounds can also comprise a neodymium iron oxide (NdFe ⁇ 3) or a dysprosium iron oxide (DyFeOj).
  • NdFe ⁇ 3 neodymium iron oxide
  • DyFeOj dysprosium iron oxide
  • elements that are useful in magnetic resonance imaging include gadolinium, terbium, tin, iron, or isotopes thereof.
  • An image can be generated in a method of the invention by computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS) image, magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emision computed tomography (SPECT), or bioluminescence imaging (BLI) or equivalent.
  • Computer assisted tomography (CAT) and computerized axial tomography (CAT) systems and devices well known in the art can be utilized in the practice of the present invention. ( See, for example, U.S. Patent Nos. 6,151,377; 5,946,371; 5,446,799; 5,406,479; 5,208,581; 5,109,397).
  • the invention may also utilize animal imaging modalities, such as MicroCAT.TM. (ImTek, Inc.).
  • Magnetic resonance imaging (MRI) systems and devices well known in the art can be utilized in the practice of the present invention.
  • a static magnetic field is applied to a tissue or a body in order to define an equilibrium axis of magnetic alignment in a region of interest.
  • a radio frequency field is then applied to the region in a direction orthogonal to the static magnetic field direction to excite magnetic resonance in the region.
  • the resulting radio frequency signals are then detected and processed, and the exciting radio frequency field is applied.
  • the resulting signals are detected by radio-frequency coils that are placed adjacent to the tissue or area of the body of interest.
  • MRI and supporting devices are commercially available for example, from Bruker Medical GMBH; Caprius; Esaote Biomedica; Fonar; GE Medical Systems (GEMS); Hitachi Medical Systems America; I ⁇ termagnetics General Corporation; Lunar Corp.; MagneVu; Marconi Medicals; Philips Medical Systems; Shimadzu; Siemens; Toshiba America Medical Systems; including imaging systems, by, e.g., Silicon Graphics.
  • the invention may also utilize animal imaging modalities such as micro-MRIs.
  • Positron emission tomography imaging (PET) systems and devices well known in the art can be utilized in the practice of the present invention.
  • a method of the invention may use the system designated Pet VI located at Brookhaven National Laboratory.
  • Pet VI located at Brookhaven National Laboratory.
  • Animal imaging modalities such as micro-PETs (Corcorde Microsystems, Inc.) can also be used in the invention.
  • Single-photon emission computed tomography (SPECT) systems and devices well known in the art can be utilized in the practice of the present invention.
  • SPECT single-photon emission computed tomography
  • the methods of the invention may also utilize animal imaging modalities, such as micro-SPECTs.
  • Bioluminescence imaging includes bioluminescence, fluorescence or chemiluminescence or other photon detection systems and devices that are capable of detecting bioluminescence, fluorescence or chemiluminescence.
  • Sensitive photon detection systems can be used to detect bioluminescent and fluorescent proteins externally; see, for example, Contag (2000) Neoplasia 2:41-52; Zhang (1994) Clin. Exp. Metastasis 12:87-92.
  • the methods of the invention can be practiced using any such photon detection device, or variation or equivalent thereof, or in conjunction with any known photon detection methodology, including visual imaging.
  • an intensified charge-coupled device (ICCD) camera coupled to an image processor may be used in the present invention.
  • ICCD intensified charge-coupled device
  • kits for carrying out the methods of the invention.
  • the kits include an antibody or an antibody fragment which binds specifically to an epitope of a kallikrein, and means for detecting binding of the antibody to its epitope associated with tumor cells, either as concentrates (including lyophilized compositions), which may be further diluted prior to use or at the concentration of use, where the vials may include one or more dosages.
  • concentrates including lyophilized compositions
  • the vials may include one or more dosages.
  • single dosages may be provided in sterilized containers, having the desired amount and concentration of agents.
  • Containers that provide a formulation for direct use usually do not require other reagents, as for example, where the kit contains a radiolabelled antibody preparation for in vivo imaging.
  • kits may also comprise instructional material teaching methodologies, e.g., how and when to administer the pharmaceutical compositions, how to apply the compositions and methods of the invention to imaging systems, e.g., computer assisted tomography (CAT), magnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI), positron emission tomography (PET), single-photon emission computed tomography (SPECT) or bioluminescence imaging (BLI).
  • Kits containing pharmaceutical compositions e.g., chimeric polypeptides, vectors, nucleic acids
  • Kits containing pharmaceutical compositions may also include directions as to indications, dosages, routes and methods of administration, and the like.
  • the immunohistochemical method of avidin-biotin complex was performed on 4 ⁇ m thick sections, using two hK6-specific antibodies, one rabbit polyclonal and one mouse monoclonal. Both antibodies were raised in- house against full length hK6, produced recombinantly in a mammalian stable cell line system (Little et al. 1997). The recombinant hK6 protein was purified by HPLC, as previously described (Little et al. 1997). The specificity of the antibodies was evaluated during development of the immunofluorometric assay, as previously described and by Western blot analysis (Diamandis et al. 2000c).
  • TRIS-buffered saline TRIS-buffered saline
  • biotinylated secondary antibody After washing with TBS, we incubated with the avidin peroxidase complex for 10 min.
  • a negative control section was always included in which the primary antibody was substituted with non-immune rabbit or mouse serum.
  • immunoabsorbtion of the primary hK6 antibody was performed by mixing it for lh with excess recombinant hK6, before immunostaining.
  • hK6 immunoreactivity using the polyclonal and the monoclonal antibody was generally localized in the cytoplasm. Both antibodies revealed the same distribution of the antigen in all tissues. Replacement of the primary antibody with non-immune serum or immunoabsorbtion of the primary antibody abolished the immunostaining in all examined tissues.
  • a supranuclear cytoplasmic, brush border and cilia staining was revealed in the fallopian tubes. Characteristic was the positivity in the premordial follicles of the ovary. In the placenta the protein was localized in the endothelia, in calcifications of the villi, as well as in "X" cells and focally, in trophoblastic cells. Genitourinary tract
  • hK6 had the same immunostaining pattern in the benign prostate gland, as hK2 and hK3.
  • the urinary tubuli of the kidney showed an immunoexpression as well.
  • the antrum of the stomach showed a focal cytoplasmic, mainly subnuclear and brush border staining; the body showed a brush border and parietal cell expression.
  • a strong positivity in foci of intestinal metaplasia in the gastric mucosa was the rule.
  • the ductal epithelium of the eosophageal glands expressed the antigen as well.
  • An expression by neuroendocrine cells throughout the gastrointestinal tract was generally obvious.
  • the reactivity by basal cells in the epithelium of the esophagus and the anus was considered to refer to neuroendocrine cells. Strong positivity was found in the cells of the islets of Langerhans in the pancreas.
  • the acinar cells of the exocrine pancreas was negative. Only some scattered positive cells were observed between them.
  • the epithelium of the medium sized pancreatic ducts showed a cytoplasmic and mainly brush border immunostaining. Hepatocytes were negative. A cytoplasmic and brush border immunostaining was observed in the bile ducts and the gallbladder mucosa. Respiratory tract
  • a focal protein immunoexpression was revealed by follicular cells, mainly in hyperplastic conditions and in oxyphilic cell metaplasia. Parathyroids An immunoexpression by the oxyphilic cells was noticed.
  • mesenchymal tissues except nervous tissue, were negative. Some weak positivity was observed in some myoepithelial cell in the wall of small arteries and by chondrocytes. Diffuse neuroendocrine system
  • hK6 Neuroendocrine cells in several organs expressed hK6.
  • Glandular epithelia constitute the main hK6 immunoexpression sites, with representative organs being breast, prostate, kidney, endometrium, colon, appendix, salivary ducts, bile ducts and gallbladder.
  • the small intestine, the stomach, the endocervix, the fallopian tube, the epididymis, the bronchus, and the upper respiratory tract show focal expression.
  • Choroid plexus epithelium, peripheral nerves and neuroendocrine cells express the protein strongly and diffusely.
  • the kallikrein 6 gene (KLK6) is one of the recently cloned members of the human tissue kallikrein gene family (Diamandis et al.2000a).
  • Messenger RNA encoding for hK6 protein (zyme/protease M/neurosin) has been previously detected in many tissues, including breast, brain, spinal cord, cerebellum, kidney, uterus, salivary gland, thymus, spleen, and testis in some mammalian species but not in mice, rats, or hamsters (Little et al. 1997; Anisowicz et al. 1996; Yamashiro et al. 1997; Yousef et al. 1999).
  • KLK6 mRNA in mature oligodendrocytes found KLK6 mRNA in mature oligodendrocytes and suggested that this protease may be important in the processes occurring after maturation of oligodendrocytes, such as myelination or turnover of the proteins in the myelin (Yamanaka et al. 1999). Little et al. detected positive immunostaining in monkey cortex cells lining the perimeter of cortical microvessels, in human brains of patients with Alzheimer's disease and in microglial cells, indicating a role of this protease in brain disease (Little et al. 1997). hK6 was also localized in the choroid plexus epithelium and correlated with Alzheimer's disease. Yamashiro et al.
  • hK6 is a cell of the diffuse neuroendocrine system and in nerves.
  • the staining was relatively strong and it could be representative of neuroendocrine differentiation.
  • the expression of hK6 is reminiscent of CD56 (natural killer cell associated antigen, neural cell adhesion molecule) and CD57 (Leu-7, T-cell surface marker) which are sensitive but not specific for cells and neoplasms with neuroendocrine differentiation (Kaufmann et al. 1997).
  • Strong hK6 expression was found in the islets of Langerhans and in the epithelium of the pancreatic ducts, but not in the acinar cells of the exocrine pancreas. It is possible that hK6 may be involved in islet hormone processing, a property already attributed to many other proteolytic enzymes (Seidah and Chretien 1999).
  • hK3 PSA
  • hK2 hK2
  • Serum hK6 concentration has also been found to be increased in ovarian carcinoma (Diamandis et al. 2000d). Some other kallikrein genes are either underexpressed or overexpressed in certain carcinomas (Yousef and Diamandis 2001).
  • the encoded proteases may serve to positively or negatively regulate cell growth or differentiation by cleavage of cell surface receptors (Coughlin 1999) or cell growth regulatory and angiogenic proteins, and by activation of other proteases for invasion and metastasis.
  • Histologic classification was based on the World Health Organization and FIGO recommendations. Of the tumors included in this study, 80 were classified as serous papillary, 32 as undifferentiated, 27 as endometrioid, 13 as mucinous, 14 as clear cell, 10 as mullerian and 4 as other.
  • the size of the residual tumors ranged from 0 to 9 cm, with a median of 1.1 cm.
  • Tumor tissue was frozen in liquid nitrogen immediately after surgery and stored at -80°C until extraction. 20 to 100 mg of frozen tissue was pulverized on dry ice to a fine powder and added to 10 volumes of extraction buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 10g/L of NP-40 surfactant, 1 mM phenylmethyl sulphonyl fluoride, lg/L of aprotinin, lg/L of leupeptin).
  • extraction buffer 50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 10g/L of NP-40 surfactant, 1 mM phenylmethyl sulphonyl fluoride, lg/L of aprotinin, lg/L of leupeptin).
  • the resulting suspension was incubated on ice for 30 minutes during which time it was vortexed every ten minutes.
  • the mixture was then centrifuged at 14,000 rpm at 4°C for 30 minutes and the supernatant (cell extract) was collected and stored at - 80°C until analysis.
  • Protein concentration of the extract was determined with the bicinchoninic acid method, with albumin as standard (Pierce Chemical Co., Rockford, IL).
  • hK6 in Ovarian Cell Extracts The concentration of hK6 in tumor cell extract was quantified with a highly sensitive and specific non-competitive immunoassay for hK6 that has been previously described and evaluated in detail (Diamandis, E. P., Yousef, G. M., Soosaipillai, A. R., Grass, L., Porter, A., Little, S., and Sotiropoulou G. Immunofluorometric assay of human kallikrein 6 (Zyme/Protease M/Neurosin) and preliminary clinical applications. Clin. Biochem., 33: 369-375, 2000).
  • the assay incorporated two hK6-specific polyclonal antibodies, one raised in mouse and the other in rabbit, in a sequential two site immunometric format with time resolved fluorescence detection. Analysis of standards, tumor cell extracts and control pools was carried out in duplicate in 96-well polystyrene microtiter plates with 200 ⁇ L of specimen added to the immunoassay. The standard curve using recombinant hK6 protein ranged from 0.5 ⁇ g/L to 200 ⁇ g/L. Assay precision was better than 10%. Signal detection and data reduction were performed automatically by the CyberFluor 615 Immunoanalyzer. Localization of hK6 in Ovarian Tumor Specimens by Immunohistochemistry.
  • a rabbit polyclonal antibody was raised against hK6 full-size recombinant protein, produced in yeast cells. Immunohistochemical staining for hK6 was performed according to a standard immunoperoxidase method. Briefly, paraffin-embedded tissue sections (4 ⁇ m) were fixed and dewaxed. Endogenous peroxidase activity was blocked with 3% aqueous hydrogen peroxide for 15 minutes. Sections were then treated with 0.4% pepsin at pH 2.0 for 5 minutes at 42°C and blocked with 20% protein blocker (Signet Labs) for 10 minutes. The primary antibody was then added at 1:400 dilution for 1 hour at room temperature.
  • biotinylated anti-rabbit antibody (Signet) was added, diluted 4-fold in antibody dilution buffer (DAKO).
  • streptavidin tagged horseradish peroxidase was added for 30 minutes at room temperature.
  • detection was achieved with amino ethyl carbazole (AEC) for 5-10 minutes.
  • AEC amino ethyl carbazole
  • Statistical analysis was performed with SPSS software (SPSS Inc. Richmond, CA). To analyze data, patients were divided into different groups according to clinical and pathological parameters. Because the distribution of hK6 mass per mg total protein (i.e. specific activity) in the ovarian tumor extracts was not Gaussian, the non-parametric Mann- Whitney U test was used to determine differences between two groups and the non-parametric Kruskal-Wallis test was used for the analysis of differences among more than two groups. These tests treated hK6 specific activity in the tumor extract (ng hK6/mg total protein) as a continuous variable.
  • hK6 tumor extract specific activity was also classified as either hK6-positive (> 35 ng/mg total protein; see Figure IB for explanation) or hK6-negative ( ⁇ 35 ng/mg total protein).
  • the relationship of this dichotomous variable to other clinicopathological correlates was established with the Chi Square ( ⁇ 2 ) test or the Fisher's Exact Test, as appropriate.
  • the impact of tumor extract hK6 specific activity on patient survival and on progression of the disease (progression-free survival) was assessed with the hazards ratio calculated by both univariate and multivariate Cox proportional hazards regression models (Cox, D. R. Regression tables and life tables. J. R. Stat. Soc. B, 34: 187-202, 1972).
  • hK6 specific activity in tumor extracts was treated both as a continuous variable and as a dichotomous variable ( ⁇ 35 ng/mg total protein, > 35 ng/mg total protein) in the analyses that follow.
  • hK6 specific activity (ng hK6/mg total protein) was significantly elevated (P ⁇ 0.001 by the Kruskal Wallis test) in extracts of ovarian tumor (mean 32.7, standard error 3.8, range 0.04 to 497) compared to extracts prepared from normal ovarian tissues (mean 3.5, standard error 2.5, range 0.05 to 20.8) or from ovarian tissue with benign disease (mean 3.2, standard error 2.6, range 0.03 to 21.5) (Figure 2). Further analysis showed there was no significant difference in hK6 specific activity among the ovarian tumors when they were stratified by histotype (i.e. serous vs undifferentiated vs endometrioid, etc).
  • hK6 status had no such effect among patients with Grade III tumor, nor could any discernible effect be demonstrated among patients with early stage disease and among those with greater than 1 cm of tumor remaining following surgery.
  • Univariate analysis revealed a 2-fold increase in risk of disease progression and of death in the subgroup of patients with advanced disease (stage III and IV) who were hK6 positive, but the effect was lost in the multivariate analysis. The opposite occurred in the subset of patients characterized by optimal debulking of the tumor at the time of surgery (remaining tumor less than 1 cm in diameter).
  • hK6 positivity had no demonstrable adverse effect on disease progression or on survival by univariate analysis, but did become statistically significant, giving a 3.5 and 5.5-fold increase in adverse risk, respectively, when the data were subjected to multivariate analysis.
  • the emergence of effects in the multivariate model when none are generated by the univariate model happens when the adjusted variables have no impact at all on the outcome. In the case here, this means that stage of disease, tumor grade, tumor histology and patient age had no prognostic potential on disease progression and overall survival in this particular subset of patients. Kaplan-Meier survival curves of the subset of patients with grade I or II ovarian tumor are shown in Figure 5.
  • hK6 positivity across the entire patient population under study was associated with about a 2-fold increase in the risk of both disease progression and of death. This effect was lost when outcomes were adjusted for the other clinicopathological variables and age in multivariate analysis of the entire patient population, but not when the multivariate analysis was restricted to those patients with lower grade tumor and with less residual tumor remaining after surgery ( ⁇ 1 cm in diameter).
  • hK6 positivity predicted about a 4-fold increase in the risk of disease progression and of death (P ⁇ 0.03) while corresponding hazard ratios in the latter subgroup were 3.75 and 5.5, respectively (P ⁇ 0.02).
  • the data show that hK6 positivity has independent predictive potential in these two subgroups and gives insight into tumor behavior over time that cannot be gleaned from the clinical parameters and pathological correlates conventionally measured. Hence hK6 testing could contribute to more individualized effective treatment of such patients.
  • hK6 is frequently overexpressed in ovarian tumors compared to nonmalignant ovarian tissue. This overexpression tended to be higher in tumors from late stage disease than from early stage disease.
  • the histochemical studies suggest that hK6 is synthesized by the epithelial cells of the ovary and is distributed diffusely within the cytoplasmic compartment.
  • Epithelial ovarian cancer has one of the worst prognoses among gynecologic malignancies, largely because over three-quarters of the diagnoses are made at a time when the disease has already established regional or distant metastases (Gatta, G., Lasota, M. B., and Verdecchia, A. Eur. J. Cancer, 34: 2218-2225, 19). Compounding the problem, tumor progression and aggressiveness correlate variably with conventional clinical and pathological markers. Thus there is an important need for additional diagnostic and prognostic markers for this disease and a number of potential markers have been identified.
  • Endometrium C droplets
  • BB endometrium
  • Thyroid gland Focal mainly in hyperplasia
  • b Fisher's Exact Test c CTX; chemotherapy, NC; no change, PD; progressive disease, CR; complete response, PR; partial response, NE; not evaluated, x. Status unknown.
  • Histologic type c 0.95 0.82-1.11 0.57 1.04 0.86-1.26 0.68
  • stage I-II hK6 univariate 0.90 0.18-4.35 0.89 1.49 0.13-16.53 0.74 hK6 multivariate" 1 1.83 0.17-19.41 0.61 2.23 0.20-25.04 0.51 stage ⁇ -rv hK6 univariate 2.04 1.26-3.29 0.004 1.98 1.12-3.47 0.017 hK6 multivariate 11 1.57 0.93-2.68 0.092 1.33 0.71-2.53 0.37
  • HK6 multivariate' 1.27 0.72-2.23 0.40 1.19 0.62-2.27 0.59 a Hazard ratio (HR) estimated from Cox proportional hazard regression model b Confidence interval of the estimated HR.
  • HR Hazard ratio
  • c Multivariate models were adjusted for stage of disease, residual tumor, histologic type and age.
  • d Multivariate models were adjusted for tumor grade, residual tumor, histologic type and age.
  • Multivariate models were adjusted for stage of disease, tumor grade, histologic type and age.
  • Diamandis EP (1999) Prostate specific antigen - its usefulness in clinical medicine. Trends Endocrinol Metab 25:14-16 Diamandis EP, Yousef GM, Luo LY, Magklara A, Obiezu CV (2000a) The new human kallikrein gene family: implications in carcinogenesis. Trends Endocrinol Metab 11:54-60
  • Diamandis EP Yousef GM, Soosaipillai AR, Bunting P (2000d) Human kallikrein 6 (zyme/protease M/neurosin): a new serum biomarker of ovarian carcinoma. Clin Biochem 33:579-583
  • Diamandis EP Yousef GM, Petraki C, Soosaipillai AR (2000e) Human kallikrein 6 as a biomarker of Alzheimer's disease. Clin Biochem 33:663-667.

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Abstract

L'invention concerne la localisation de troubles associés aux kallikréines, plus particulièrement le cancer de l'ovaire, par des agents ayant été construits pour cibler les kallikréines, de préférence la kallikréine 6 et facultativement d'autres kallikréines.
PCT/CA2002/001556 2001-10-16 2002-10-16 Localisation de troubles associes aux kallikreines WO2003034068A1 (fr)

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WO2004029285A3 (fr) * 2002-09-26 2004-08-19 Mount Sinai Hospital Corp Methodes de detection du cancer de l'appareil endocrinien
WO2004075713A3 (fr) * 2003-02-26 2004-10-28 Mount Sinai Hospital Corp Dosage a marqueurs multiples utilise pour depister un cancer des ovaires
WO2005075634A1 (fr) * 2004-01-28 2005-08-18 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques des maladies associees au kallikrein 12 (klk12)
WO2016061142A1 (fr) 2014-10-14 2016-04-21 Novartis Ag Molécules d'anticorps de pd-l1 et leurs utilisations

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WO1998011238A2 (fr) * 1996-09-13 1998-03-19 Dana-Farber Cancer Institute Protease m, une nouvelle serine protease
WO2001059158A1 (fr) * 2000-02-11 2001-08-16 The Board Of Trustees Of The University Of Arkansas Compositions et methodes permettant un diagnostic precoce du cancer ovarien
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004029285A3 (fr) * 2002-09-26 2004-08-19 Mount Sinai Hospital Corp Methodes de detection du cancer de l'appareil endocrinien
WO2004075713A3 (fr) * 2003-02-26 2004-10-28 Mount Sinai Hospital Corp Dosage a marqueurs multiples utilise pour depister un cancer des ovaires
WO2005075634A1 (fr) * 2004-01-28 2005-08-18 Bayer Healthcare Ag Methodes diagnostiques et therapeutiques des maladies associees au kallikrein 12 (klk12)
WO2016061142A1 (fr) 2014-10-14 2016-04-21 Novartis Ag Molécules d'anticorps de pd-l1 et leurs utilisations
EP4245376A2 (fr) 2014-10-14 2023-09-20 Novartis AG Molécules d'anticorps de pd-l1 et leurs utilisations

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