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WO2012004565A1 - Biomarqueur brf1 du cancer - Google Patents

Biomarqueur brf1 du cancer Download PDF

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Publication number
WO2012004565A1
WO2012004565A1 PCT/GB2011/001024 GB2011001024W WO2012004565A1 WO 2012004565 A1 WO2012004565 A1 WO 2012004565A1 GB 2011001024 W GB2011001024 W GB 2011001024W WO 2012004565 A1 WO2012004565 A1 WO 2012004565A1
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WO
WIPO (PCT)
Prior art keywords
brfl
cancer
individual
sample
expression
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PCT/GB2011/001024
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English (en)
Inventor
Robert White
Hing Leung
Original Assignee
Robert White
Hing Leung
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Publication of WO2012004565A1 publication Critical patent/WO2012004565A1/fr

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Classifications

    • 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
    • G01N33/57419—Specifically defined cancers of colon
    • 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
    • G01N33/57449—Specifically defined cancers of ovaries
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • This invention relates to the identification of biomarkers for the screening, diagnosis and prognosis of cancers, such as prostate cancer .
  • Prostate cancer develops in the prostate, a gland that forms part of the male reproductive system and it is the most common cancer in men, excluding non-melanoma skin cancer.
  • Carcinoma of the prostate is generally a tumour of older men, and 95% of prostate carcinomas are adenocarcinomas. The rate at which the tumours grow varies but it tends to be slow growing. Patient survival is linked to the extent of the disease: if confined to the prostatic capsule, survival of over 5 years can be anticipated;
  • Prostate cancer may cause pain, difficulty in urinating, problems during sexual intercourse, or erectile dysfunction. Other symptoms can potentially develop during later stages of the disease. Many factors, including age, genetics and diet, have been implicated in the development of prostate cancer.
  • PSA Serum prostate specific antigen
  • DRE digital rectal examination
  • prostate cancer screening The major concern about prostate cancer screening is the potential for over-diagnosis of prostate cancers that would be unlikely to pose a threat for morbidity or mortality.
  • the PSA "cut-point" above which further evaluation to rule out prostate cancer (by prostate biopsy) should be recommended is a controversial issue.
  • the prostate also frequently manifests benign enlargement (benign prostatic hyperplasia (BPH) and chronic or recurrent inflammation (prostatitis). Like prostate cancer, each of these conditions can elevate PSA.
  • BPH benign prostatic hyperplasia
  • prostatitis chronic or recurrent inflammation
  • Diagnosis of prostate cancer ultimately requires needle biopsy specimens and microscopic analysis of prostate tissue, which can be graded histologically, via the assignment of a Gleason grade or score (that predicts the behaviour of prostate cancer) .
  • Prostate cancers can be difficult to recognize and difficult to distinguish from other prostate abnormalities such as prostate intraepithelial neoplasia ("PIN”) .
  • PIN prostate intraepithelial neoplasia
  • Most experienced prostate pathologists use a combination of findings to make a diagnosis of prostate cancer based on needle biopsy, such as:
  • basal epithelial cell markers such as cytokeratins K5 and K14 - these can be used to help distinguish benign from
  • profiling can aid in prostate cancer diagnosis
  • High-grade PIN is a likely precursor to prostate cancer and is a lesion characterized by the proliferation of malignant-appearing prostate epithelial cells within the confines of otherwise normal glandular structures.
  • HG-PIN is identified in about 5% of men subjected to prostate biopsies. About 96 percent of all men diagnosed with prostate cancer survive at least five years, and 75 percent survive at least 10 years. Many men diagnosed will undergo watchful waiting regimes as opposed to active treatment. The most common treatment options for prostate cancer are surgery or hormonal therapy, depending on the staging at diagnosis. However, the percentage of cancer patients that relapse remains high (44-64%). Treatment is costly and harsh, and there is a need to identify the patients that would benefit from treatment.
  • the present inventors have recognised that expression of the transcription factor Brfl is closely associated with cancer, for example prostate cancer, and in particular with malignant cancers (e.g. prostate cancers with a Gleason score of 8 or more or a
  • Brfl may therefore be useful in screening, diagnosis and prognosis of cancers, such as prostate cancer .
  • One aspect of the invention provides a method of assessing an individual for cancer comprising;
  • the presence of Brfl in the sample obtained from the individual may be indicative of said individual having or being at risk of having cancer.
  • the presence of Brfl in the sample may be indicative that the individual has cancer while the absence of one or more cells expressing Brfl in the sample may be indicative that the individual does not have cancer.
  • expression of Brfl may be increased relative to controls, such as normal non-cancer cells.
  • cancer may include any type of solid cancer or malignant lymphoma and especially leukaemia, sarcomas, skin cancer, bladder cancer, breast cancer, uterus cancer, testicular cancer, ovary cancer, prostate cancer, lung cancer, colon and colorectal cancer, cervical cancer, liver cancer, head and neck cancer, oesophageal cancer, pancreas cancer, renal cancer, stomach cancer and cerebral cancer.
  • Preferred cancers include prostate cancer, ovarian cancer, colon cancer and testicular cancer.
  • a cancer as described herein is a prostate cancer.
  • prostate cancer and non-cancer conditions such as prostate intraepithelial neoplasia and benign prostatic hyperplasia, may be distinguished.
  • Methods as described herein may be useful in distinguishing between benign cancer and malignant or aggressive cancer.
  • benign prostate cancer and malignant or aggressive prostate cancer may be distinguished. This may inform decisions about future treatment regimens .
  • the presence of one or more cells expressing Brfl in the sample obtained from the individual may be indicative of said individual having aggressive or malignant cancer, for example a prostate cancer with a Gleason score of 4, 5, 6, 7, 8 or more; or a Gleason grade of 3 or more.
  • An individual identified as having a malignant or aggressive cancer as described herein may be subjected to further treatment, for example by surgery or hormonal therapy, in accordance with standard medical practice.
  • An individual identified as having a non- malignant or benign cancer may be subjected to mild or no further treatment and/or increased monitoring.
  • Brfl may therefore be useful as a stratification marker to identify patients who require increased monitoring or treatment.
  • the expression of Brfl may be useful in determining the prognosis of a cancer in an individual and the likely progress of the condition and the risk of morbidity and/or mortality.
  • the presence of one or more cells expressing Brfl in the sample may be indicative that the individual has a poor prognosis and a high risk of mortality and the absence of one or more cells expressing Brfl in the sample may be indicative that the individual has a good prognosis and a low risk of mortality.
  • An individual may be healthy or asymptomatic or may be suspected of or at risk of suffering from cancer, for example prostate cancer.
  • an individual may be suffering from cancer and/or may have one or more symptoms of cancer, for example prostate cancer .
  • the individual may have been previously diagnosed as having cancer or suspected of having cancer by one or more diagnostic tests.
  • the individual may have been previously diagnosed as having prostate cancer or suspected of having prostate cancer by one or more prostate cancer tests, such as serum PSA testing; digital rectal examination (DRE) ; and/or immunohistochemical staining for AMACR or basal epithelial cell markers, such as cytokeratins K5 and K14.
  • the individual may be undergoing treatment for cancer and methods of the invention may be useful in determining the progress of the cancer treatment.
  • a method may further comprise determining the level, presence or expression of one or more other cancer markers.
  • a method of assessing prostate cancer may further comprise determining the level, presence or expression of one or more other prostate cancer markers, such as PSA, AMACR or basal epithelial cell markers, such as cytokeratins K5 and K14.
  • a sample obtained from an individual preferably comprises one or more cells from the individual, preferably prostate cells.
  • the sample may be a tissue sample, for example a biopsy from a cancerous tissue as described above, in particular a needle biopsy, or, in certain contexts, a non-cancerous tissue.
  • the presence or amount of Brfl in one or more cells in the sample may be determined.
  • a sample may be a sample of body fluid, such as saliva, cerebrospinal fluid, blood, urine or semen.
  • body fluid such as saliva, cerebrospinal fluid, blood, urine or semen.
  • a sample of urine may be collected following digital rectal examination to dislodge cells into the urinary tract.
  • Brfl is a 90 kD polypeptide that is an essential component of the transcription factor TFIII B. It binds directly to RNA polymerase III and serves to recruit it to promoters.
  • transcription factor plays a central role in transcription
  • Brfl (GenelD 2972) may have the amino acid sequence of Genbank accession numbers of NP_001510.2 GI : 22035556 (SEQ ID NO: 2) or NP_663718.1 GI : 22035558 (SEQ ID NO: 4) or may be a variant thereof.
  • a variant Brfl polypeptide may comprise an amino acid sequence which has one or more allelic variations, for example, deletions, insertions or substitutions of one or more amino acids, relative to the wild-type Brfl sequence set out above.
  • a variant polypeptide may have one, two, three, four or more mutations relative to the wild-type sequence.
  • a variant Brfl polypeptide may have at least 80%, at least 85%, at least 90%, at least 95% at least 98% or at least 99% sequence identity with a Brfl polypeptide sequence as set out above.
  • Brfl may be encoded by the nucleotide sequence of NM_001519.2 GI : 22035555 ⁇ SEQ ID NO: 1) or NM__145685.2 GI : 148833509 (SEQ ID NO:3) or a variant thereof.
  • a variant Brfl nucleic acid may comprise a nucleotide sequence which has one or more allelic variations, for example, deletions, insertions or substitutions of one or more nucleotides, relative to the wild-type Brfl nucleotide sequence, as set out above.
  • a variant nucleic acid may have one, two, three, four or more mutations relative to the wild-type sequence.
  • a variant nucleic acid may have at least 80%, at least 85%, at least 901, at least 95%, at least 98% or at least 99% sequence identity with a Brfl nucleotide sequence as set out above.
  • allelic variant of a reference Brfl amino acid sequence of a biomarker may differ from a reference sequence by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30 or 30-50 amino acids.
  • An allelic variant of a reference Brfl nucleotide sequence may differ from a reference nucleotide sequence of a biomarker by insertion, addition, substitution or deletion of 1 nucleotide, 2, 3, 4, 5-10, 10-20 20-30 or 30-50 nucleotides .
  • a method of assessing an individual for cancer may thus comprise; providing a sample of cells obtained from the individual, and; determining the presence in the sample of one or more cells expressing Brfl and/or the number or proportion of cells in the sample of expressing Brfl.
  • the presence, number or proportion of cells expressing Brfl may be indicative of said individual having or being at risk of having cancer.
  • the presence, number or proportion of cells expressing Brfl may be indicative of the prognosis of the cancer in the individual i.e. the presence of one or more such cells may be indicative of that the cancer is aggressive or malignant cancer.
  • a method of assessing an individual for prostate cancer may comprise; providing a sample of cells obtained from the individual, and; determining the presence in the sample of one or more cells expressing Brfl and/or the number or proportion of cells in the sample expressing Brfl.
  • the presence, number or proportion of cells expressing Brfl may be indicative of said individual having or being at risk of having prostate cancer.
  • the presence, amount or proportion of cells expressing Brfl may be indicative of the prognosis of the prostate cancer in the individual i.e. the presence, amount or proportion of cells expressing Brfl may be indicative that the prostate cancer is aggressive or malignant prostate cancer.
  • the expression of Brfl may be determined at the protein level by determining the presence or amount of Brfl polypeptide in the sample. For practical purposes, or at least commercial purposes bearing in mind cost and time, assessment of target protein expression at the protein level may be preferred over assessment at the nucleic acid level.
  • a method of determining the presence, absence or level of Brfl polypeptide in a sample from an individual may include contacting the sample with a specific binding member which specifically binds to the Brfl polypeptide, and determining binding of the specific binding member to the sample. Binding of the specific binding member to the sample may be indicative of the presence or amount of Brfl within the sample.
  • an antibody or other binding member which specifically binds to an antigen such as Brfl may not show any significant binding to molecules other than the antigen.
  • an antibody may specifically bind to a particular epitope which is carried by a number of antigens, in which case the antibody will be able to bind to the various antigens carrying the epitope.
  • Samples to be subjected to a contact with a binding member in accordance with methods of the invention may be prepared using any available technique which allows binding of a specific binding molecule to the Brfl polypeptide. Various techniques are standard in the art.
  • Binding may be detected using any method or detectable label described herein. This, and any other binding detection method described herein, may be interpreted directly by the person performing the method, for instance, by visually observing a detectable label. Alternatively, this method, or any other binding detection method described herein, may produce a report in the form of an autoradiograph, photograph, computer printout, flow cytometry report, graph, chart, test tube, container or well containing the result, or any other visual or physical representation of a result of the method.
  • the amount of binding of the binding member to Brfl in the sample may be determined. Screening for Brfl binding and/or the
  • quantitation thereof may be useful, for instance, in screening patients for cancers and/or any other disease or disorder involving increased Brfl expression and/or activity.
  • a diagnostic or prognostic method may comprise (i) obtaining a tumour, tissue or fluid sample from a patient, (ii) exposing said tumour, tissue or fluid sample to one or more binding members which bind Brfl; and (iii) detecting the amount of binding of the binding member to the sample, as compared with a control sample, wherein an increased amount of binding as compared with controls may indicate increased or elevated Brfl expression in the sample.
  • the reactivities of a binding member such as an antibody on normal and test samples may be determined by any appropriate means.
  • the reporter molecules may directly or indirectly generate detectable, and preferably measurable, signals.
  • the linkage of reporter molecules may be directly or indirectly, covalently, e.g. via a peptide bond or non-covalently . Linkage via a peptide bond may be as a result of recombinant expression of a gene fusion encoding binding molecule (e.g. antibody) and reporter molecule.
  • fluorochromes include fluorescein, rhodamine, phycoerythrin and Texas Red.
  • Suitable chromogenic dyes include diaminobenzidine .
  • Other reporters include macromolecular colloidal particles or particulate material such as latex beads that are coloured, magnetic or paramagnetic, and biologically or chemically active agents that can directly or indirectly cause detectable signals to be visually observed, electronically detected or otherwise recorded.
  • These molecules may be enzymes that catalyse reactions that develop or change colours or cause changes in electrical properties, for example. They may be molecularly excitable, such that electronic transitions between energy states result in characteristic spectral absorptions or emissions. They may include chemical entities used in conjunction with biosensors. Biotin/avidin or biotin/streptavidin and alkaline phosphatase detection systems may be employed. Further examples are horseradish peroxidase and chemiluminescence .
  • the mode of determining binding is not a feature of the present invention and those skilled in the art are able to choose a suitable mode according to their preference and general knowledge.
  • Many different assay formats suitable for detecting Brfl are known in the art, including non-competitive and competitive assays and immunoassays. Suitable approaches include immunohistochemical staining, immunocytochemical staining, Western Blotting,
  • an assay for Brfl in a sample may involve the use of a capture binding member with binds Brfl and a detection binding member which either binds to a different epitope on Brfl than the first binding member or which binds to the capture binding member.
  • Suitable Brfl binding members may be produced using routine techniques or obtained from commercial sources (e.g. Novus
  • the capture binding member may be immobilised on a solid substrate and the detection binding member may be non-immobilised.
  • Preferred binding members for use in aspects of the present invention include antibodies and fragments or derivatives thereof ( ⁇ antibody molecules' ) .
  • Antibody molecules are well known in the art and are described in more detail below.
  • a binding member may be immobilised to the solid support in a number of different ways known in the art.
  • the binding member may be adsorbed directly to the solid support, e.g. through
  • the binding member may be covalently attached to the solid support.
  • the solid support may be chemically modified to introduce or activate
  • the binding member may be indirectly attached to the solid support by a specific binding interaction, for example by an interaction between biotin and avidin, or by immobilising protein A or protein G to the solid support followed by specific binding to the binding member itself.
  • the capture binding member which binds Brfl may be immobilised at a solid support.
  • the immobilised binding member may then be brought into contact with a sample of interest. If the sample contains Brfl, it will bind with the immobilised binding member.
  • the detection binding member which binds Brfl is then added and allowed to bind.
  • the capture binding member is not immobilised and the detection binding member may be a binding member which binds Brfl at a different epitope from the capture binding member or a binding member which binds to the capture binding member.
  • the second binding member may be labelled with a detectable label.
  • binding of the second binding member may be determined using a third binding member, such as an antibody, which specifically binds to the second binding member and is labelled with a detectable label.
  • a third binding member such as an antibody
  • Brfl in the sample may be bound by the capture binding member, and immobilised at the support.
  • Second binding member binds to the captured Brfl and is itself immobilised at the support.
  • labelled third binding member may bind to the second binding member and also be immobilised at the support.
  • Brfl in the sample may be bound by the capture binding member.
  • Second binding member binds to the captured Brfl or the capture antibody and is itself detected via a label or labelled third binding member binds to the second binding member and is detected.
  • the third binding member may, for example, directly bind to the second binding member (e.g. an anti-IgG antibody) or may bind to an affinity tag which is linked or fused to the second binding member.
  • Suitable affinity tags include biotin or peptidyl sequences, such as MRGS(H) 6 , DYKDDDDK (FLAGTM), T7-, S- ( KETAAAKFERQHMDS ) , poly-Arg
  • Tag.100 Qiagen; 12 aa tag derived from mammalian MAP kinase 2
  • Cruz tag 09TM MKAEFRRQESDR, Santa Cruz Biotechnology Inc.
  • Cruz tag 22TM MRDALDRLDRLA, Santa Cruz Biotechnology Inc.
  • the amount of labelled second or third binding member bound directly or indirectly to the solid support is then measured, whereby the amount of labelled binding member detected is directly proportional to the amount of Brfl in the sample.
  • a method of measuring Brfl in a sample may comprise; contacting the sample with a capture binding member with binds Brfl, and;
  • the expression of Brfl may be determined at the nucleotide level by determining the presence or amount of Brfl nucleic acid, for example mRNA, encoding the Brfl polypeptide in the sample .
  • determining the presence or absence in a sample of a Brfl nucleic acid sequence and may for example include oligonucleotide probe binding, DNA amplification, for example by PCR, or microarray hybridisation. All of these approaches are well known in the art.
  • Another aspect of the present invention provides for a method of categorising a sample from an individual as (i) normal, (ii) potentially or actually pre-cancerous or cancerous, dysplastic, or neoplastic and benign or (iii) cancerous and malignant, the method including determining binding to a sample of a specific binding member directed against a Brfl polypeptide. The pattern or degree of binding may be compared with that for a known normal sample and/or a known abnormal sample.
  • a method may be used to characterise a cell, for example a cancer cell, as malignant, for example in the prognosis of a prostate cancer.
  • binding of (e.g.) an anti- Brfl specific binding member to a sample provides for categorising the tissue from which the sample is derived as potentially or actually pre-cancerous or cancerous, benign, dysplastic or neoplastic or cancerous and potentially malignant and metastatic.
  • the method may be used to pre screen samples before further analysis or testing.
  • the method may also be useful for screening or analysis of samples previously tested using another prostate cancer diagnostic techniques.
  • a specific binding molecule which binds Brfl may be provided in a kit, which may include instructions for use in accordance with a method of the invention and methods for interpreting and analyzing the data resulting from the performance of the assay.
  • the binding member may b labelled to allow its reactivity in a sample to be determined, e.g. as described further below. Further, the binding member may or may not be attached to a solid support.
  • kits are provided as a further aspect of the invention.
  • One or more other reagents may be included, such as labelling molecules, and so on (see below) .
  • the reagents may include a second, different binding member which binds to the first specific binding member and is conjugated to a
  • Antibody-based kits may also comprise beads for conducting an immunoprecipitation .
  • Reagents may be provided within containers, which protect them from the external environment, such as a sealed vial.
  • a kit may include one or more articles for providing or preparing the test sample itself, depending on the tissue of interest.
  • a kit may include any combination of, or all of, a blocking agent to decrease non-specific staining, a storage buffer for preserving binding molecule activity during storage, staining buffer and/or washing buffer to be used during antibody staining, a positive control, a negative control and so on.
  • Positive and negative controls may be used to validate the activity and correct usage of reagents employed in accordance with the invention and which may be provided in a kit.
  • Controls may include samples, such as tissue sections, cells fixed on coverslips and so on, known to be either positive or negative for the presence of Brfl. The design and use of controls is standard and well within the routine
  • Methods of assessing a cancer condition as described herein may be employed before, during and/or after a course of prostate cancer treatment in order to determine the progress and/or effectiveness of the treatment; to determine the most appropriate treatment or level of monitoring for the individual.
  • Antibody molecules and other binding members that bind specifically to Brfl polypeptide may be useful both in the screening diagnosis and prognosis of prostate cancer. Antibodies may also be useful in the treatment of prostate cancer.
  • Antibodies that are specific for a Brfl polypeptide may be obtained using techniques that are standard in the art. Methods of producing antibodies include immunising a mammal (e.g. mouse, rat, rabbit, horse, goat, sheep, monkey or bird such as chicken) with the protein or a fragment thereof, or a cell or virus that expresses the protein or fragment. Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to Brfl. For instance, Western blotting techniques or immunoprecipitation may be used (Armitage et al, 1992, Nature 357: 80-82) . The production of specific monoclonal antibodies is also well established in the art.
  • a mammal e.g. mouse, rat, rabbit, horse, goat, sheep, monkey or bird such as chicken
  • Antibodies may be obtained from immunised animals using any of a variety of techniques known in the art, and screened, preferably using binding of antibody to Brf
  • an antibody specific for a target may be obtained from a recombinantly produced library of expressed immunoglobulin variable domains, e.g. using lambda bacteriophage or filamentous
  • the library may be naive, that is constructed from sequences obtained from an organism which has not been immunised with the target or may be one constructed using sequences obtained from an organism which has been exposed to the antigen of interest (or a fragment thereof) .
  • Antibodies may be modified in a number of ways. Indeed, unless context precludes otherwise, the term “antibody” should be construed as covering any specific binding substance having an antibody antigen-binding domain. Thus, this covers antibody fragments, derivatives, and functional equivalents, including any polypeptide comprising an immunoglobulin binding domain, whether natural or synthetic. Chimaeric molecules comprising an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore included. Cloning and expression of chimaeric antibodies are described in EP-A-0120694 and EP-A-0125023.
  • Example antibody fragments capable of binding an antigen or other binding partner are the Fab fragment consisting of the VL, VH, CI and CHI domains; the Fd fragment consisting of the VH and CHI domains; the Fv fragment consisting of the VL and VH domains of a single arm of an antibody; the dAb fragment which consists of a VH domain; isolated CDR regions and F(ab')2 fragments, a bivalent fragment including two Fab fragments linked by a disulphide bridge at the hinge region. Single chain Fv fragments are also included. Recombinant expression of polypeptides, including antibody
  • an anti-Brfl antibody may be useful in the treatment of cancer, such as prostate cancer.
  • an antibody molecule which is specific for Brfl may be conjugated or bound to a cytotoxic agent. Binding of the antibody molecule to the Brfl may be used to selectively target the cytotoxic agent to a cancer cells.
  • cytotoxic agents are known in the art.
  • Determination of binding to Brfl polypeptide in vivo may be used to identify localisations of cancer cells in the body. Labelled binding members against Brfl may be administered to an individual and binding within the body determined. When a radionucleotide such as Iodine-125, Indium-Ill, Thallium-201 or Technetium-99m is attached to an antibody or other binding members, and the antibody or other binding member localises preferentially in tumour rather than normal tissues, the presence of radiolabel in tumour tissue can be detected and quantitated using a gamma camera or scintigraphy. Radiolabelling with technetium-99m is described in Pak et al (1992), Nucl . Med. Biol. 19; 699-677.
  • prognosis of an individual having cancer such as prostate cancer.
  • Another aspect of the invention provides a method of determining the malignancy of a cancer in an individual, the method comprising,
  • the presence or amount of Brfl polypeptide or nucleic acid may be determined in the sample.
  • the presence of Brfl nucleic acid or polypeptide may be determined as described above.
  • Brfl expression may be indicative that the cancer is malignant (e.g. a prostate cancer having a Gleason grade of 3 or more and/or a Gleason score of 5, 6, 7, 8 or more).
  • the absence of Brfl expression or the absence of Brfl above a threshold value relative to controls may be indicative that the cancer is benign (e.g. it has a Gleason score of 4 or less and/or a Gleason grade of less than 3) .
  • Another aspect of the invention provides a method for assessing the responsiveness of a cancer in an individual to treatment may comprise :
  • the level or amount of Brfl may be measured in a first sample obtained from the individual before said administration as described above and in a second sample obtained from the individual after said administration. A difference, for example a decrease, between the level or amount of Brflin the first and second samples, may be indicative that the disease condition is responsive to said
  • Another aspect of the invention provides a method for assessing the responsiveness of a cancer in an individual to treatment comprising:
  • a reduction in the expression of Brfl following administration of the cancer therapy may be indicative of the efficacy of the therapy in the individual.
  • a method for monitoring the treatment of cancer in individual may comprise :
  • the method may further comprise;
  • the method may further comprise;
  • a maximal change in the level or amount of Brfl which is sustained during the treatment is indicative that the altered regimen is effective for treating the cancer in the individual.
  • Measurement of Brfl may also be useful in identifying a cohort of patients for clinical trials.
  • a method for identifying a cohort of patients may comprise:
  • the identified cohort of patients may all have high or low levels of Brfl (i.e. levels above or below the threshold value).
  • Suitable treatments which may be monitored and/or assessed in a patient as described above are well known in the art and include cytotoxic regimes of drugs, such as topoisomerases (e.g. topotecan, irinotecan, rubitecan), alkylating agents (e.g. DTIC, temozolamide ) and platinum based drugs (e.g. carboplatin, cisplatin) and/or ionising radiation.
  • topoisomerases e.g. topotecan, irinotecan, rubitecan
  • alkylating agents e.g. DTIC, temozolamide
  • platinum based drugs e.g. carboplatin, cisplatin
  • Another aspect of the invention provides a method of treating cancer, such as prostate cancer, in an individual, the method comprising inhibiting the activity of Brfl polypeptide in one or more cells of said individual.
  • the activity or function of Brfl may be inhibited, for example, by administering an antagonist of Brfl to said individual.
  • a suitable antagonist may be an antibody molecule, peptide or small organic molecule .
  • An antagonist which acts on Brfl may be specifically targeted to a tumour site using conventional techniques.
  • Suitable Brfl antagonists include antibody molecules as described above.
  • An alternative approach to inhibition employs regulation at the nucleic acid level to inhibit activity or function by down- regulating production of Brfl.
  • the activity of Brfl polypeptide may be inhibited by reducing or abolishing expression of Brfl
  • polypeptide using anti-sense or RNAi technology.
  • Anti-sense oligonucleotides may be designed to hybridise to the complementary sequence of nucleic acid, pre-mRNA or mature mRNA, interfering with the production of Brfl polypeptide so that its expression is reduced or completely or substantially completely prevented.
  • anti-sense techniques may be used to target control sequences of a gene, e.g. in the 5' flanking sequence, whereby the antisense oligonucleotides can interfere with expression control sequences.
  • the construction of antisense sequences and their use is described for example in Peyman and Ulman, Chemical Reviews, 90:543-584, (1990) and Crooke, Ann. Rev. Pharmacol. Toxicol., 32:329-376, (1992).
  • Oligonucleotides may be generated in vitro or ex vivo for
  • administration or anti-sense RNA may be generated in vivo within cells in which down-regulation is desired.
  • double-stranded DNA may be placed under the control of a promoter in a "reverse
  • RNA orientation such that transcription of the anti-sense strand of the DNA yields RNA which is complementary to normal mRNA transcribed from the sense strand of the target gene.
  • the complementary anti- sense RNA sequence is thought then to bind with mRNA to form a duplex, inhibiting translation of the endogenous mRNA from the target gene into protein. Whether or not this is the actual mode of action is still uncertain. However, it is established fact that the technique works .
  • the complete sequence corresponding to the coding sequence in reverse orientation need not be used. For example fragments of sufficient length may be used. It is a routine matter for the person skilled in the art to screen fragments of various sizes and from various parts of the coding or flanking sequences of a gene to optimise the level of anti-sense inhibition. It may be advantageous to include the initiating methionine ATG codon, and perhaps one or more nucleotides upstream of the initiating codon.
  • a suitable fragment may have about 14-23 nucleotides, e.g. about 15,
  • anti-sense is to use a copy of all or part of the target gene inserted in sense, that is the same, orientation as the target gene, to achieve reduction in expression of the target gene by co-suppression; Angell & Baulcombe (1997) The EMBO Journal
  • Double stranded RNA has been found to be even more effective in gene silencing than both sense or antisense strands alone (Fire
  • RNA interference is a two-step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs ( ⁇ 2nt) . The siRNAs target the corresponding mRNA sequence
  • RNAi may be also be efficiently induced using chemically synthesized siRNA duplexes of the same structure with 3 '-overhang ends (Zamore PD et al. Cell, 101, 25-33, (2000)). Synthetic siRNA duplexes have been shown to specifically suppress expression of endogenous and heterologeous genes in a wide range of mammalian cell lines
  • nucleic acid is used which on
  • ribozyme able to cut nucleic acid at a specific site - thus also useful in influencing gene expression.
  • Background references for ribozymes include Kashani-Sabet and
  • an inhibitor of Brfl activity may comprise a nucleic acid molecule comprising all or part of the or wild type Brfl coding sequence or the complement thereof
  • Such a molecule may suppress the expression of the Brfl polypeptide and may comprise a sense or anti-sense Brfl coding sequence or may be a Brfl specific ribozyme, according to the type of suppression to be employed.
  • the type of suppression will also determine whether the molecule is double or single stranded and whether it is RNA or DNA.
  • administration is preferably in a "prophylactically effective amount” or a "therapeutically effective amount” (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual.
  • a prophylaxis may be considered therapy
  • the actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors .
  • a Brfl antagonist may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.
  • compositions may include, in addition to active ingredient, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • a pharmaceutically acceptable excipient such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material will depend on the route of administration, which may be oral, or by injection, e.g. cutaneous, subcutaneous or intravenous.
  • compositions for oral administration may be in tablet, capsule, powder or liquid form.
  • a tablet may include a solid carrier such as gelatin or an adjuvant.
  • Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
  • the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability.
  • isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, or Lactated Ringer's Injection.
  • Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
  • Figure 1 shows immunohistochemical (ICH) analysis with a polyclonal antibody against Brfl on biopsy material from 149 patients with prostate cancer and 21 patients with benign prostate hyperplasia. Brfl levels are elevated significantly (P ⁇ 0.001) in the cancer samples .
  • Figure 2 shows with a polyclonal antibody against Brfl on biopsy material from 21 patients with benign prostate hyperplasia and 149 patients with prostate cancer, split according to stage.
  • Figure 3 shows nuclear Brfl levels are predictive of survival in the 149 patients with prostate cancer analysed.
  • Figure 4 shows immunohistochemical analysis of Brfl expression in normal prostate (upper) and a prostate cancer (lower) .
  • Figure 5 shows immunohistochemical analysis of Brfl expression in a prostate tissue section, where normal tissue (left) can be seen adjacent to tumour (right) .
  • Figure 6 shows immunohistochemical analysis of Brfl (lower) and Ki67 (upper) expression in a prostate cancer section (Gleason grade 4).
  • Figure 7 shows immunohistochemical analysis of Brfl expression in sections of benign prostate hyperplasia (upper) and Gleason grade 5 prostate cancer (lower) .
  • Figure 8 shows immunohistochemical analysis of Brfl expression in sections of normal tissue (A and C) and tumour (B and D) from human testis (A and B) or ovary (C and D) .
  • Figure 9 shows immunohistochemical analysis of Brfl expression in colonic crypt cells before and 4 days after induction of colon carcinogenesis by cre-mediated deletion of the APC tumour
  • a polyclonal antibody against Brfl was raised and used in immuno- histochemistry (IHC) on biopsy material of a cohort of 149 patients with prostate cancer and 21 with benign prostate hyperplasia (BPH) .
  • the cohort of 149 patients had different grades of carcinoma and were all untreated.
  • Brfl was observed to be significantly increased (P ⁇ 0.001) in the cancer samples ( Figure 1).
  • the survival curves were derived from the same 149 patients and are shown in Figure 3. Patients with low Brfl staining have a median survival of 3402 days, whereas the median is only 875 days for the patients with high Brfl staining. Ki67 and Brfl staining are not well correlated, showing that Brfl is not simply an indicator of proliferation .
  • Ki67 is a marker of cell proliferation (figure 6). It is clear that Brfl staining is not restricted to cells that stain positively for Ki67 (black) . This indicates that Brf1 elevation in cancers is not merely a consequence of cell proliferation. Brfl is therefore over- expressed in prostate cancers independently of cell proliferation

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Abstract

La présente invention concerne le facteur de transcription Brf1 et son utilisation en tant que biomarqueur dans le diagnostic et le pronostic du cancer.
PCT/GB2011/001024 2010-07-06 2011-07-06 Biomarqueur brf1 du cancer WO2012004565A1 (fr)

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US9885718B2 (en) 2014-07-02 2018-02-06 Dragon Victory Development Ltd. Specific biomarker set for non-invasive diagnosis of liver cancer

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9506925B2 (en) 2014-07-02 2016-11-29 Dragon Victory Development Ltd. Specific biomarker set for non-invasive diagnosis of liver cancer
US9885718B2 (en) 2014-07-02 2018-02-06 Dragon Victory Development Ltd. Specific biomarker set for non-invasive diagnosis of liver cancer
US10620209B2 (en) 2014-07-02 2020-04-14 Dragon Victory Development Ltd. Specific biomarker set for non-invasive diagnosis of liver cancer

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