EP1432821A2 - Cancer - Google Patents

Cancer

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Publication number
EP1432821A2
EP1432821A2 EP02765065A EP02765065A EP1432821A2 EP 1432821 A2 EP1432821 A2 EP 1432821A2 EP 02765065 A EP02765065 A EP 02765065A EP 02765065 A EP02765065 A EP 02765065A EP 1432821 A2 EP1432821 A2 EP 1432821A2
Authority
EP
European Patent Office
Prior art keywords
duttl
cells
cancer
population
binding agent
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP02765065A
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German (de)
English (en)
Inventor
Pamela Rabbitts
T.H. MRC Lab. of Molecular Biology RABBITTS
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Medical Research Council
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Medical Research Council
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Filing date
Publication date
Priority claimed from GB0123630A external-priority patent/GB0123630D0/en
Priority claimed from GB0127668A external-priority patent/GB0127668D0/en
Application filed by Medical Research Council filed Critical Medical Research Council
Publication of EP1432821A2 publication Critical patent/EP1432821A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily

Definitions

  • the present invention relates to methods for the diagnosis, imaging and treatment of cancer cells.
  • it relates to the use of a tumour related molecule which is present at a higher density on cancer cells than normally dividing cells in the diagnosis, imaging, prophylaxis and treatment of cancer.
  • tumour suppressor genes TSG
  • TSG tumour suppressor genes
  • the successful identification of these genes has frequently relied on the isolation of candidate TSG from within much smaller homozygously deleted regions, mapping within the larger region of allele loss (1).
  • Various deletion mapping studies have indicated that several distinct regions within the chromosomal 3 loci are involved in the onset and/or progression of lung cancer. Recent studies include a recent extensive study of 151 lung tumour biopsies and cell lines with 28 markers of polymorphic loci (2) on chromosome 3. Using FISH, homozygous deletions at 3 l2, 3pl4 and 3p21 have been shown to also exist in biopsy material (4).
  • tumour suppressor molecules may be useful in the prophylaxis and/or treatment of tumours.
  • Cancers may be detected by treating samples of tissues with agents known to bind to defined types of cancer cell. Such detection generally requires removal of tissue samples from a vertebrate. Suitable agents include antibodies and the like.
  • Some cancers can be detected by imaging: radiological methods(e.g. X-rays) are the most familiar. Although valuable, there are problems of selectivity-i.e. other non-malignant nodules are detected and sensitivity-i.e. very small clusters of tumour cells e.g. micrometasteses are undetectable.
  • radiological methods e.g. X-rays
  • selectivity-i.e. other non-malignant nodules are detected and sensitivity-i.e. very small clusters of tumour cells e.g. micrometasteses are undetectable.
  • tumour -specific antigens were sought but generally have not been found in solid tumours. It is becoming apparent that tumour targets need not be expressed exclusively by tumours to be of value. What is required is that the tumour over-expresses a cell surface antigen compared to surrounding tissue.
  • DUTT1 (deleted in TJ2020) was found to map within smaller homozygous deletions in two other tumour cell lines (9).
  • the DUTTl/ROBOl gene is widely expressed in mammals and codes for a receptor with a domain structure of the NCAM family (13).
  • Several lung tumour cell lines including NIH-H219X have previously been shown to possess one or more deletions within the gene encoding DUTTl, therefore implicating DUTTl in lung cancer.
  • the present inventors set out to overcome the problems of the prior art. In particular, they set out to identify one or more molecules which play a role in the onset or progression of lung tumour formation. In addition they set out to establish a method for the imaging, diagnosis, prophylaxis and/or treatment of cancer cells.
  • the present inventors have surprisingly found that the level of the tumour suppressor protein DUTTl is high in cancer cells, including carcinoma insitu (pre-invasive cancer cells), and is low on non-cancerous epithelium, for instance bronchial epithelium.
  • negligible protein is detected on epithelial stem cells.
  • the present invention provides a method for the early detection of cancer in a population of cells comprising the steps of:
  • the present invention provides a method for the early detection of cancer in a population of cells comprising the steps of : ( 1 ) providing a population of cells
  • step 3 (3) obtaining a population of cells for diagnosis, (4) assaying the cell population of step 3, for an increased level of DUTTl in any one or more of those cells when compared with the reference level of DUTTl.
  • the present invention provides a method for the diagnosis of cancer in a population of cells comprising the steps of: (l)providing a population of cells
  • the present invention provides a method for the diagnosis of cancer in a population of cells comprising the steps of :
  • step (3) obtaining a population of cells for diagnosis, (4) assaying the cell population of step 3, for an increased level of DUTTl in any one or more of those cells when compared with the reference level of DUTTl.
  • 'cancer' as herein defined include the ability of a cell to undergo endless replication, loss of contact inhibition, invasiveness and the ability to metastasize. That is, when the cell divides in an uncontrollable way and can not recognise its own natural boundary, the cancer cells obtain the ability to spread to other areas of the body. Mutations within the nucleic acid of one or more cells are involved in the onset of cancer. Often, more than one nucleic acid mutation or other aberrant cellular event is required for the development of tumours (bundles of aberrantly dividing cells), that is tumour formation is a multi-signal event.
  • cancer cells include any cells which exhibit any one or more of the following features aberrant cell division, aberrant contact inhibition, aberrant cell differentiation as compared with cells behaving normally within their native environment, the ability of the cell to invade tissues, and the ability to metastasise.
  • the definition of 'cancer cells' in the context of the present invention therefore includes within its scope tumour cells and also cells prior to the formation of tumours in so far as they possess one or more of the requisite characteristics listed above.
  • cancer cells according to the present invention includes metastatic cells.
  • the method of the present invention is suitable for the diagnosis and/or detection of many forms of cancer.
  • the cancer may be one or more selected from the group consisting of: epithelial cancer, sarcoma and lymphoma.
  • the cancer is lung cancer, advantageously it is human lung cancer.
  • the cells for diagnosis include bronchial epithelial cells and/or bronchial hyperplasia of epithelium, and/or cells derived from lymph nodes
  • the method is an in vitro method.
  • the term the 'early detection' of cancer means the detection of cancer prior to the onset of one or more clinical signs of cancer in a patient.
  • Clinical signs of cancer will be known to those skilled in the art and includes the formation of tumours and metastases. The early detection of cancer
  • steps (1) and (3) may be the same population.
  • steps (1) to (4) may be performed simultaneously, or separately. Alternatively, several steps may be performed simultaneously, and others separately.
  • a reference level of DUTTl may be established using one or more agents selected from the group consisting of: anti-DUTTl antibodies, DUTTl binding peptides and small molecules which bind to DUTTl.
  • agents selected from the group consisting of: anti-DUTTl antibodies, DUTTl binding peptides and small molecules which bind to DUTTl.
  • DUTTl antibodies are used. Suitable methods for measuring DUTTl levels using these agents will be familiar to one skilled in the art and are described herein.
  • DUTTl binding agents may be bound to a population of cells, and a reference level of DUTTl established by directly comparing cancerous and non-cancerous cells within the same cell population. This has the advantage that protein levels in cancer cells and surrounding normally dividing cells can be compared easily and simultaneous, and that quantitative measurements of DUTTl levels do not need to be made. Cells binding an increased level of DUTTl binding agent, compared with the normal cells will be easily distinguishable.
  • the DUTTl binding agents will comprise or have associated with them means for their detection. Suitable means may be naturally occurring or synthetic molecules and will be familiar to those skilled in the art, and are described herein.
  • the present invention provides, the use of a DUTTl binding agent in the diagnosis of cancer.
  • the cancer may be one or more selected from the group consisting of: epithelial cancer, sarcoma and lymphoma.
  • the cancer is lung cancer, advantageously it is human lung cancer.
  • the cells for diagnosis include bronchial epithelial cells and/or bronchial hyperplasia of epithelium, and/or cells derived from lymph nodes
  • the method is an in vitro method.
  • DUTTl binding agents for diagnosis are as herein described,.
  • the DUTTl binding agent is an antibody raised against DUTTl .
  • DUTTl binding agents can be used as a method of imaging or visualising cancer cells.
  • the present invention provides a method for the selective labelling of cancer cells comprising the step of treating one or more cancer cells with a DUTTl binding agent.
  • the present invention provides the use of a DUTTl binding agent in the selective labelling of cancer cells.
  • Cells suitable for selective labelling include any cell which expresses DUTTl, preferably at high levels. Suitable cells include epithelial cancer cells, sarcoma cells and lymphoma cells.
  • the cells are lung cancer cells, preferably human lung cancer cells.
  • the cancer cells are metastatic cells and/or primary cancer cells.
  • the method is for the selective labelling of cancer cells selected from the group consisting of: human cancer cells, human epithelial cancer cells, human sarcoma cells, human haematopoietic cells.
  • Suitable DUTTl binding agents are as described herein.
  • the term 'labelling' in the context of the present invention means the selective binding of an agent to cells, in this case to cancer cells and not to normally dividing cells.
  • the labelling agent comprises or has associated with it means permitting the detection of the label.
  • Suitable means include fluorescent, phosphorescent, or radio-active agents as herein described.
  • Suitable means for detection of the labelled cells will vary according to whether the labelled cells are to be detected in vitro or in vivo. Suitable in vitro methods include but are not limited to autoradiography and fluorescence or phosphorescence detection. Suitable, in vivo methods include but are not limited to nuclear magnetic resonance, autoradiography and so on. Those skilled will be aware of other suitable methods.
  • the method of this aspect of the present invention may be used for the in vivo imaging of cancer cells, preferably metastatic cancer cells.
  • the method is used for the imaging of human cancer cells.
  • the method is used for the imaging of human cancer cells, which may be primary cancer cells or metastatic cells within the body of a patient.
  • Suitable DUTTl binding agents include any one or more selected from the group consisting of the following: anti-DUTTl antibodies, DUTTl binding peptides and small molecules which bind to DUTTl.
  • DUTTl antibodies are used.
  • the present invention provides a method for the imaging of cancer cells comprising the step of treating one or more cancer cells with one or more DUTTl binding agent/s wherein the DUTTl binding agent further comprises detection means.
  • the term 'imaging' refers to the generation of a 2 dimensional or 3 dimensional picture/image of the cancer cells.
  • the imaging may allow the relative position of the cancer cells within the in vivo environment, preferably the human body to be established. Due to the high density of DUTTl found on lung cancer cells including pre-invasive cancer cells, a high signal to noise ratio can be achieved, allowing high sensitivity imaging. Such a high signal to noise ratio is particularly important in the detection of small bundles of cancer cells, that is metastatic cells.
  • this method is also of use in the imaging of primary cancer cells.
  • the method is for the in vivo imaging of cancer cells. More preferably it is for the imaging of human cancer cells.
  • the method is for the imaging of cancer cells selected from the group consisting of: human epithelial cancer cells, human sarcoma cells, human haematopoietic cancer cells.
  • the present invention provides a composition comprising DUTTl , or a binding agent thereof.
  • the present invention provides a method for the intemalisation of one or more DUTTl binding agents comprising the step of treating a cell with one or more DUTTl binding agents.
  • 'treating' refers to the process of bringing one or more cells into contact with a DUTTl binding agent. Suitable methods for 'treating' cells will depend upon whether the procedure is carried out in vivo or in vitro and are described herein. In a preferred embodiment of this aspect of the invention, the method occurs in vivo.
  • the term 'intemalisation' means bringing one or more molecules into the interior of the cell.
  • it describes bringing one or more molecules into the cytoplasm of a cell.
  • at least one molecule is a DUTTl binding agent.
  • Suitable DUTTl binding agents are herein described.
  • Cells suitable for treatment with a DUTTl binding agent according to the method of the present invention includes any cell which expresses DUTTl.
  • Suitable cells include epithelial cells. Those skilled in the art will be aware of other suitable cell types.
  • DUTTl binding agents are herein described.
  • the DUTTl binding agent is a antibody raised against DUTTl.
  • it is a monoclonal antibody.
  • the present invention provides the use of DUTTl, and/or a binding agent thereof in the preparation of a medicament for the prophylaxis or treatment of cancer.
  • the use is in the treatment of lung cancer. In a further embodiment of this aspect of the invention, the use is in the treatment of epithelial cancer. In a further embodiment still, the use is in the treatment of sarcoma. In a further preferred embodiment, the use is in the treatment if lymphoma.
  • Fig. 1 Targeted mutation of the Duttl/Robol gene .A, the genomic structure of the targeted locus and the targeting construct and mutant allele.
  • the filled box represents exon 2.
  • Fragments a and b are the hybridisation probes used in (B) below. The outer dashed lines indicate the fragment used for homologous recombination. E EcoRI, S Sad, B BamHI.
  • B Following transfection of the targeting construct into ES cells, Sacl digested DNA was prepared from G418 resistant ES(+/-) and control ES cells(+/+) and analysed by Southern blot analysis The filter was hybridised to probe
  • Fig. 2 Duttl/Robol protein analysis (24). A, Western blotting analysis of protein isolated from the organs shown from wild type (+/+) and Duttl/Robol ' ⁇ (-/-) day 15 embryos using antiserum raised against a C-terminal DUTT1/ROBO1 peptide.
  • C detection by immunohistochemistry of Duttl/Robol protein using anti-peptide antiserum in (A) in bronchial epithelium with Cy-3-TSA amplification system (NEN Life Sciences) applied to the same section as in (B) x200 magnification.
  • Fig. 3 Gross morphological phenotypes of wild-type and Duttl/Robol ' ' ' mice. A wild ttyyppee mmoouussee aanndd iittss lluunnggss sshhoowwn below (+/+) compared to a Duttl/Robol ' ' mouse and its lungs shown below (-/-).
  • FIG. 4 Histological analysis of lungs of Duttl/Robol " mice and wild-type littermates. Paraformaldehyde fixed 4 micrometre sections of lung tissue were H&E stained and photographed.
  • A wild-type lung, embryo day El 5.5 x 400 magnification
  • B Duttl/Robol '1' lung, embryo day 15.5, x 400 magnification
  • C wild-type newborn lung, x 100 magnification
  • D Duttl/Robol '1' newborn lung x 100 magnification (arrow indicates bronchi)
  • E wild-type adult lung x 100 magnification, F, Duttl/Robol '1' adult lung, x 100 magnification, G, wild-type adult lung at x 400 magnification, H, Duttl/Robol "1' adult lung, at x 400 magnification, I, Duttl/Robol '1' adult lung, at x 400 magnification, focal dysplasia indicated by arrow
  • Fig 5. Detection by immunohistochemistry of DUTTl/ROBOl protein using anti-peptide antibody in a section of formalin fixed human squamous cell carcinoma of the lung .
  • 'cancer' includes the ability of the cancer cell to undergo endless replication, loss of contact inhibition, invasiveness and the ability to metastasise. That is, when the cell divides in an uncontrollable way and can not recognise its own natural boundary, the cancer cells obtain the ability to spread to other areas of the body. Mutations within the nucleic acid of one or more cells are involved in the onset of cancer. Often, more than one nucleic acid mutation or other aberrant cellular event is required for the development of tumours (bundles of aberrantly dividing cells), that is tumour formation is a multi-signal event.
  • cancer cells include any cells which exhibit any one or more of the following features aberrant cell division, aberrant contact inhibition, aberrant cell differentiation as compared with cells behaving normally within their native environment, the ability of the cell to invade tissues, and the ability to metastasise.
  • the definition of 'cancer cells' in the context of the present invention therefore includes within its scope tumour cells and also cells prior to the formation of tumours in so far as they possess one or more of the requisite characteristics listed above.
  • cancer cells according to the present invention includes metastatic cells.
  • a 'tumour suppressor molecule' is a molecule one function of which is to suppress tumourigenesis. Certain cancers have been found to be associated with mutant suppressor genes for example p53 and RB. However as indicated above often more than one abherent cell component or signal is required to initiate and/or to cause the progression of cancer.
  • Bronchial epithelial hyperplasia The airway of the lung in descending order of size are bronchi, bronchioles and alveoli. Bronchial refers to the first two. .
  • the lung consists of mesenchymal cells and epithelial cells which line the airways. Hyperplasia means over growth.
  • the epithelium is normally a sheet of cells one layer thick but when it becomes hyperplastic it thickens and becomes disorderly. Bronchial epithelial hyperplasia.
  • Specific labelling of a cell' in the context of the present invention means the selective/specific binding of a labelling agent to a cell. That is, that when a labelling agent is exposed to a cell population, only those cells showing certain characteristics will bind to the labelling agent. Generally, the characteristics include the presence of certain cell surface features, for example the presence of a surface antigen which binds to the labelling agent.
  • An 'Antibody' for example IgG, IgM, IgA, IgD or IgE
  • fragment such as a FAb, F(Ab') 2 , Fv, disulphide linked Fv, scFv, diabody
  • the present invention provides a method for the early diagnosis of cancer in a population of cells comprising the steps of:
  • the present invention provides a method for the early diagnosis of cancer in a population of cells comprising the steps of : (1 )providing a population of cells
  • step 3 assaying the cell population of step 3, for an increased level of DUTTl in any one or more of those cells when compared with the reference level of DUTTl.
  • the present invention provides a method for the diagnosis of cancer in a population of cells comprising the steps of: (l)providing a population of cells
  • the present invention provides a method for the diagnosis of cancer in a population of cells comprising the steps of : (l)providing a population of cells
  • Cell samples are obtained from a vertebrate for treatment using methods familiar to those skilled in the art.
  • the animals may be sacrificed, and the tissue for diagnosis extracted.
  • Tissue slices may be prepared using methods familiar to those skilled in the art.
  • cells for diagnosis may be obtained by performing a biopsy (removal of a small amount of tissue from a vertebrate, which is preferably alive).
  • DUTTl binding agents may be bound to a population of cells, and a reference level of DUTTl established by directly comparing cancerous and non- cancerous cells within the same cell population. This has the advantage that protein levels in cancer cells and surrounding normally dividing cells can be compared easily and simultaneous, and that quantitative measurements of DUTTl levels do not need to be made.
  • Antibodies raised against DUTTl, and DUTTl peptides may be prepared using standard laboratory techniques. Either recombinant proteins or those derived from natural sources can be used to generate antibodies. For example, the protein (or "immunogen") is administered to challenge a mammal such as a monkey, goat, rabbit or mouse. The resulting antibodies can be collected as polyclonal sera, or antibody- producing cells from the challenged animal can be immortalized (e.g. by fusion with an immortalizing fusion partner to produce a hybridoma), which cells then produce monoclonal antibodies.
  • the antigen protein is either used alone or conjugated to a conventional carrier in order to increases its immunogenicity, and an antiserum to the peptide-carrier conjugate is raised in an animal, as described above.
  • Coupling of a peptide to a carrier protein and immunizations may be performed as described (Dymecki et al. (1992) J. Biol. Chem., 267: 4815).
  • the serum is titered against protein antigen by ELISA or alternatively by dot or spot blotting (Boersma and Van Leeuwen (1994) J. Neurosci. Methods, 51: 317).
  • the serum is shown to react strongly with the appropriate peptides by ELISA, for example, following the procedures of Green et al. (1982) Cell, 28: 477.
  • monoclonal antibodies may be prepared using any candidate antigen, preferably bound to a carrier, as described by Arnheiter et al. (1981) Nature, 294, 278.
  • Monoclonal antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from animals into which the hybridoma tissue was introduced. Nevertheless, monoclonal antibodies may be described as being “raised against” or “induced by” a protein. After being raised, monoclonal antibodies are tested for function and specificity by any of a number of means. Similar procedures can also be used to test recombinant antibodies produced by phage display or other in vitro selection technologies.
  • Monoclonal antibody-producing hybridomas can be screened for antibody binding to the immunogen, as well.
  • Particularly preferred immunological tests include enzyme-linked immunoassays (ELISA), immunoblotting and immunoprecipitation (see Voller, (1978) Diagnostic Horizons, 2: 1, Microbiological Associates Quarterly Publication, Walkersville, MD; Voller et al. (1978) J. Clin. Pathol., 31: 507; U.S. Reissue Pat. No. 31,006; UK Patent 2,019,408; Butler (1981) Methods Enzymol., 73: 482; Maggio, E.
  • ELISA enzyme-linked immunoassays
  • Northern and Western blotting may be performed using methods familiar to those skilled in the art and detailed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (1999).
  • Tissue samples may be prepared using methods familiar to those skilled in the art and DUTTl detected using any of the reagents referred to above.
  • An example of a typical protocol is detailed below:
  • Tissues or whole embryos are fixed in 4% paraformaldehyde in PBS buffer for at least 24h, paraffin embedded and processed to give 4 mm sections. Sections were deparaffinised and endogenous peroxide quenched. After blocking, Duttl/Robol was detected using the polyclonal antisera raised against DUTTl followed by donkey biotin-conjugated anti-rabbit secondary antibody (SantaCruz USA) and amplification with the Vectastain ABC kit (Vector Laboratories USA) as per the manufacturers protocol. Positive staining was detected using nickel enhanced diaminobenzidine tetrahydrochloride (DAB) and counterstained with Fast Red. Slides were mounted with Vectashield-mounting medium. Staining was blocked by preincubation of the immunising peptide with the antisera. Sections were stained with haematoxylin and eosin (H&E).
  • H&E haematoxylin and
  • the functional portion of the DUTTl binding agent when the DUTTl binding agent, is used for diagnosis, usually comprises and may consist of a radioactive atom for scintigraphic studies, for example technetium 99m ( 99m Tc) or iodine- 123 ( I23 I).
  • a radioactive atom for scintigraphic studies for example technetium 99m ( 99m Tc) or iodine- 123 ( I23 I).
  • the present invention provides a method for the selective labelling of cancer cells comprising the step of treating one or more cancer cells with a DUTTl binding agent.
  • the term 'labelling' means the selective binding of an agent to cells, in this case to cancer cells and not to normally dividing cells.
  • the labelling agent comprises or has associated with it means permitting the detection of the label. Suitable means include fluorescent, phosphorescent, or radio-active agents, or radioopaque molecules or agents, such as metal particles, which are readily visualised within an embryo or a cell mass.
  • Suitable cancer cell, particularly lung cancer cell labelling agents include antibodies raised against DUTTl as herein defined, DUTTl binding peptides, and/or small molecule agonists which mimic the binding of the natural ligand to the receptor.
  • Suitable fluorophores are known in the art, and include chemical fluorophores and fluorescent polypeptides, attached to immunoglobulin molecules by incorporating binding sites therefor into the immunoglobulin molecule during the synthesis thereof.
  • the fluorophore is a fluorescent protein, which is advantageously GFP or a mutant thereof.
  • GFP and its mutants may be synthesised together with the immunoglobulin or target molecule by expression therewith as a fusion polypeptide, according to methods well known in the art.
  • a transcription unit may be constructed as an in-frame fusion of the desired GFP and the immunoglobulin or target, and inserted into a vector as described above, using conventional PCR cloning and ligation techniques.
  • Antibodies may be labelled with any agent capable of generating a signal.
  • the signal may be any detectable signal, such as the induction of the expression of a detectable gene product.
  • detectable gene products include bioluminescent polypeptides, such as luciferase and GFP, polypeptides detectable by specific assays, such as ⁇ -galactosidase and CAT, and polypeptides which modulate the growth characteristics of the host cell, such as enzymes required for metabolism such as HIS3, or antibiotic resistance genes such as G418.
  • the signal is detectable at the cell surface.
  • the signal may be a luminescent or fluorescent signal, which is detectable from outside the cell and allows cell sorting by FACS or other optical sorting techniques.
  • Immunosensors are biochemical detectors comprising an antigen or antibody species coupled to a signal transducer which detects the binding of the complementary species (Rabbany et ah, 1994 Crit Rev Biomed Eng 22:307-346; Morgan et al, 1996 Clin Chem 42:193-209). Examples of such complementary species include the antigen Zif 268 and the anti-Zif 268 antibody. Immunosensors produce a quantitative measure of the amount of antibody, antigen or hapten present in a complex sample such as serum or whole blood (Robinson 1991 Biosens Bioelectron 6:183-191). The sensitivity of immunosensors makes them ideal for situations requiring speed and accuracy (Rabbany et al, 1994 Crit Rev Biomed Eng 22:307-346).
  • Detection techniques employed by immunosensors include electrochemical, piezoelectric or optical detection of the immuno interaction (Ghindilis et al., 1998 Biosens Bioelectron 1:113-131).
  • An indirect immunosensor uses a separate labelled species that is detected after binding by, for example, fluorescence or luminescence (Morgan et al., 1996 Clin Chem 42:193-209).
  • Direct immunosensors detect the binding by a change in potential difference, current, resistance, mass, heat or optical properties (Morgan et al, 1996 Clin Chem 42:193-209).
  • Indirect immunosensors may encounter fewer problems due to non-specific binding (Attridge et al., 1991 Biosens Bioelecton 6:201-214; Morgan et al, 1996 Clin Chem 42:193-209). such as GFP and mutants thereof (see WO 97/28261).
  • a method for the imaging of cancer cells comprising the step of treating one or more cancer cells with one or more DUTTl binding agent/s wherein the DUTTl binding agent further comprises detection means.
  • Suitable detection means as herein defined include molecules/agents which can be readily detected when associated with, or form a component of the specific labelling agent as herein defined, when present within an in vivo environment, preferably the human body.
  • radioactive agents/molecules examples include technetium 99m ( 99m Tc) or iodine- 123 ( I). Tumours can then readily be visualised by detecting the emission of radioactive particles using methods known to those skilled in the art.
  • Detection molecules/agents such as iodine-123, iodine-313, indium-I l l, fluorine-19, carbon- 13, nitrogen- 15, oxygen- 17, gadolinium, manganese or iron allow visualisation of cancer cells using NMR. This has the advantage that a whole body scanning can be performed.
  • PET Phase Emission Tomography
  • Suitable detection means for use in PET include 11C methionine and FDG. Descriptions of procedures and protocols for using PET are familiar to those skilled in the art..
  • the present invention provides the use of DUTTl, and/or a binding agent thereof in the preparation of a medicament for the prophylaxis or treatment of cancer.
  • Therapeutic and prophylactic uses of DUTTl and/or binding agents thereof involve the administration of the above to a recipient mammal, such as a human.
  • Substantially pure DUTTl and/or binding agents thereof of at least 90 to 95% homogeneity are preferred for administration to a mammal, and 98 to 99% or more homogeneity is most preferred for pharmaceutical uses, especially when the mammal is a human.
  • the DUTTl and/or binding agents thereof may be used diagnostically or therapeutically (including extracorporeally) or in developing and performing assay procedures using methods known to those skilled in the art.
  • DUTTl and binding agents thereof may be effective in treating cancer related diseases.
  • the present invention includes the method of treating cancer related disease with an effective amount of DUTTl or DUTTl binding agents, according to the present invention.
  • the DUTTl and DUTTl binding agents of the present invention can be provided as isolated and substantially purified proteins and protein fragments in pharmaceutically acceptable compositions using formulation methods known to those of ordinary skill in the art. These compositions can be administered by standard routes.
  • ophthalmic including intravitreal or intracameral
  • nasal topical (including buccal and sublingual), intrauterine, vaginal or parenteral (including subcutaneous, intraperitoneal, intramuscular, intravenous, intradermal, intracranial, intratracheal, and epidural) transdermal, intraperitoneal, intracranial, intracerebroventricular, intracerebral, intravaginal, intrauterine, or parenteral (e.g., intravenous, intraspinal, subcutaneous or intramuscular) routes.
  • parenteral e.g., intravenous, intraspinal, subcutaneous or intramuscular routes.
  • the DUTTl and DUTTl binding agents may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the active ingredient and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • the DUTTl and DUTTl binding agents of the present invention may be incorporated into biodegradable polymers allowing for sustained release of the compound, the polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor or implanted so that the DUTTl binding agent or fragment is slowly released systemically.
  • biodegradable polymers and their use are described, for example, in detail in Brem et al (J. Neurosurg 1991 74:441-446).
  • Osmotic minipumps may also be used to provide controlled delivery of high concentrations of DUTTl or binding agents thereof, including fragments thereof through cannulae to the site of interest, such as directly into a metastatic growth or into the vascular supply to that tumor.
  • the DUTTl and DUTTl binding agents of the present invention may be linked to cytotoxic agents which are infused in a manner designed to maximize delivery to the desired location.
  • cytotoxic agents which are infused in a manner designed to maximize delivery to the desired location.
  • ricin-linked high affinity DUTTl, and/or binding agents thereof are delivered through a cannula into vessels supplying the target site or directly into the target.
  • agents are also delivered in a controlled manner through osmotic pumps coupled to infusion cannulae.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above Tecited, or an appropriate fraction thereof, of the administered ingredient. It should be understood that in addition to the ingredients, particularly mentioned above, the formulations of the present invention may include other agents conventional in the art having regard to the type of formulation in question.
  • DUTTl or binding agents thereof may be administered in any suitable way, usually parenterally, for example intravenously or intraperitoneally, in standard sterile, non- pyrogenic formulations of diluents and carriers, for example isotonic saline (when administered intravenously).
  • diluents and carriers for example isotonic saline (when administered intravenously).
  • the pro-drug is administered, usually as a single infused dose, or the tumour is imaged.
  • DUTTl or the DUTTl binding agent may be immunogenic, cyclosporin or some other immunosuppressant can be administered to provide a longer period for treatment but usually this will not be necessary.
  • the timing between administrations of DUTTl binding agents and/or fragments thereof may be optimised in a non-inventive way since tumour/normal tissue ratios of conjugate (at least following intravenous delivery) are highest after about 4-6 days, whereas at this time the absolute amount of DUTTl binding agents, or fragments of bound to the tumour, in terms of percent of injected dose per gram, is lower than at earlier times.
  • the optimum interval between administration of DUTTl binding agent and/or fragments thereof will be a compromise between peak tumour concentration of and the best distribution ratio between tumour and normal tissues.
  • the dosage of the DUTTl binding agent or fragment thereof will chosen by the physician according to the usual criteria. At least in the case of methods employing a targeted enzyme such as ⁇ -glucosidase and intravenous amygdalin as a toxic pro-drug, 1 to 50 daily doses of 0.1 to 10.0 grams per square metre of body surface area, preferably 1.0-5.0 g/m are likely to be appropriate. For oral therapy, three doses per day of 0.05 to 10. Og, preferably 1.0-5.0g, for one to fifty days may be appropriate.
  • the dosage of DUTTl binding agent or fragment thereof will similarly be chosen according to normal criteria, particularly with reference to the type, stage and location of the tumour and the weight of the patient.
  • the duration of treatment will depend in part upon the rapidity and extent of any immune reaction to the DUTTl binding agent, or fragment thereof.
  • the functional portion of DUTTl binding agent, or fragment thereof may comprise a highly radioactive atom, such as iodine-131, rhenium- 186, rhenium- 188, yttrium-90 or lead- 212, which emits enough energy to destroy neighbouring cells, or a cytotoxic chemical compound such as methotrexate, adriamicin, vinca alkaliods (vincristine, vinblastine, etoposide), daunorubicin or other intercalating agents.
  • a highly radioactive atom such as iodine-131, rhenium- 186, rhenium- 188, yttrium-90 or lead- 212, which emits enough energy to destroy neighbouring cells, or a cytotoxic chemical compound such as methotrexate, adriamicin, vinca alkaliods (vincristine, vinblastine, etoposide), daunorubicin or other intercal
  • the radio- or other detection agents may be incorporated in the DUTTl binding agent and/or fragments thereof in known ways.
  • a DUTTl binding peptide may be biosynthesised or may be synthesised by chemical amino acid synthesis using suitable amino acid precursors involving, for example, fluorine- 19 in place of hydrogen.
  • Labels such as 99m Tc, 123 I, 186 Rh, 188 Rh and m In can be attached via a cysteine residue in the peptide.
  • Yttrium-90 can be attached via a lysine residue.
  • the IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res. Commua 80: 49-57 can be used to incorporate iodine- 123. "Monoclonal Antibodies in
  • compositions comprising an effective amount of DUTTl binding agent and/or a fragment thereof can be used in the prophylaxis, suppression or treatment of cancer related disorders.
  • disorders include but not limited to: solid tumours; blood born tumours such as leukemias; tumor metastasis; benign tumours, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; rheumatoid arthritis; psoriasis; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, corneal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis; Osier-Webber Syndrome; myocardial angiogenesis; plaque neovascularization; telangiectasia; hemophiliac joints; angiofibroma; wound granulation; corornay collaterals; cerebral
  • prevention involves administration of the protective composition prior to the induction of the disease.
  • suppression refers to administration of the composition after an inductive event, but prior to the clinical appearance of the disease.
  • Treatment involves administration of the protective composition after disease symptoms become manifest.
  • a mouse brain cDNA library purchased from Stratagene (La Jolla, CA) and a mouse 13.5 day embryo cDNA library, purchased from Life Technology were screened with human DUTTl/ROBOl cDNA clones. Positive cDNA clones were sequenced and overlapped to generate a contig map that was entered into the Genbank data base. All sequencing reactions were performed using the Sanger dideoxy chain termination method using the Sequenase version 2 kit (United States Biochemical, USB). The sequence of the cDNA clones was independently confirmed (Oswell Research Products Ltd., Lab 5005, Victoria).
  • Duttl/Robor /' mice generation of Duttl/Robor /' mice (see Fig. 1).
  • An 8.0 kb Sacl fragment containing exon 2 of the mouse Duttl/Robol gene was used for construction of the targeting vector.
  • a neo expression cassette (16) (pMCl-neo poly[A]), corrected to the wild-type neo sequence (17), was used to replace a 0.7 kb genomic Hindlll, BamHI fragment spanning sequences coding for exon 2 of the Duttl gene. The replacement sequence was flanked 5' by 4.4 kb and 3' by 1.4 kb of genomic sequence.
  • a thymidine kinase gene expression cassette (18) was ligated to a unique Xbal site at the 5' of the longer homologous arm. The final vector was linearized with Hindlll for electroporation. CCB ES cells were maintained on mouse embryonic feeders.
  • Hindlll-digested targeting vector DNA Twenty-five micrograms of Hindlll-digested targeting vector DNA were used to electroporate 1 x 10 7 CCB ES cells. Sacl digested genomic DNA from cell clones surviving G418 (GIBCO) at 400 g/ml and Gancyclovir at 2.5 M selection was subjected to Southern blot analysis. Positively targeted clones were confirmed using a 5' 0.7 kb BamHI fragment internal probe and a 0.4 kb Hindlll/Sacl probe external to the homologous sequence on genomic DNA digested with Sacl. ES cells from two independent clones were used for injection into blastocysts derived from C57BL/6 mice.
  • Blastocysts were transferred to pseudo- pregnant females, and chimaeric offspring were detected by the presence of agouti colour on a non-agouti background. Chimaeric males were mated to C57BL/6 females to produce ES cell-derived offspring. Their genotype was confirmed by Southern blot analysis of tail DNA. Mice heterozygous for the gene targeting event, i.e. with a deletion of exon 2 of the Duttl/Robol gene, were inter-crossed to generate homozygotes.
  • RNA analysis Total RNA was prepared from various tissues with Trizol Reagent (GIBCO BRL). Reverse transcription reactions were performed on total RNA (5 micrograms) in 20microlitres containing 0.5 microgms of random hexamer (Pharmacia) 25 mM Tris-HCl, pH 8.3, 50 mM KC1, 2.0 mM dithiothreitol, 5.0 mM MgCl 2 , 1 mM each of dATP, dCTP, dGTP & dTTP, 1 u/microlitre of RNasin ribonuclease inhibitor (Promega, Madison, WI), and 10 u/microgm of SUPER RT (HT Biotechnology Ltd, Cambridge, England).
  • RT reaction was at 42°C for 40 minutes.
  • the product of RT was diluted five-fold and amplified by PCR. (30 cycles of lmin denaturation at 95°, lmin annealing at 55° and lmin extension at 72 0) .
  • the primers used were: exon 1 forward 5'-AGGGATTGACAAGCCTCCGG-3', exon 2 reverse 5'-AGCTACCTCCAGCGATGCGT-3', exon 3 reverse 5'- . CATCTTTATCATCCAGGGGT-3'.
  • the products were visualised following electrophoresis on agarose gels.
  • Protein lysates were prepared with a lysis buffer composed of 9 M urea, 75 mM Tris-HCl pH 7.5 and 0.15 M beta-mercaptoethanol as described (19). Protein concentration was determined using the Bradford assay, (Bio-Rad Laboratories). For Western blotting, 50 microgm of protein lysate were subjected to SDS-PAGE on a 10% polyacrylamide gel and transferred onto a nitrocellulose membrane (BioTrace; Gelman Sciences). Transfer was assessed by Ponceau S staining (Bio-Rad).
  • Filters were incubated with a polyclonal antisera raised against the C-terminal peptide of DUTTl/ROBOl (CYERGEDNNEELEETES) followed by HRP-conjugated mouse anti-rabbit IgG and visualised by enhanced chemiluminscence (Amersham, Pharmacia).
  • Example 5 Histological analysis and immunohistochemistry. Tissues or whole embryos were fixed in 4% paraformaldehyde in PBS buffer for at least 24h, paraffin embedded and processed to give 5 micrometre sections. Sections were stained with haematoxylin and eosin (H&E). For immunohistochemistry, Duttl/Robol was detected using the polyclonal antisera described above followed by donkey biotin-conjugated anti- rabbit secondary antibody and Cy-3 conjugated Streptavidin. Slides were mounted with Vectashield-mounting medium. Surfactant protein-C was detected using a commercially available antibody (SantaCruz) followed by donkey biotin-conjugated anti-goat secondary antibody and Cy-3 conjugated Streptavidin.
  • Duttl/Robol mutant mice The Duttl/Robol gene was disrupted in mice by targeted replacement of exon 2 with the neomycin phosphotransferase gene, producing a mutant form of the gene unable to code for the first immunoglobulin domain (Fig.lA). This exactly reproduces a truncated DUTTl/ROBOl transcript detected in the human lung cancer cell line NIH-H219X (9). Following transfection, DNA from G418-resistant embryonic stem cell (ES) clones was analysed by Southern blotting to identify correctly targeted clones by hybridisation with probe (a) within the targeting construct (Fig. IB) and with probe (b) external to the homologous integration.
  • ES G418-resistant embryonic stem cell
  • Example 7 Duttl/Robol protein in mutant and normal mice.
  • the resulting protein was reduced to about one half of the level detected in wild-type organs (Fig. 2A).
  • the Duttl/Robol protein is shorter by 20kDa due to loss of the first immunoglobulin domain encoded by exon 2 (Fig. 2A).
  • Immunohistochemistry was performed using a polyclonal antibody raised against the C terminus of human DUTTl/ROBOl (Clark, KJ, JX, EH and PHR unpublished observations), which recognises the mouse protein. This detected the protein at high levels in epithelial cells lining bronchi but at low or undetectable levels in adjacent mesenchyme and cells lining alveoli (Fig. 2B).
  • Duttl/Robol "7" mice frequently die at birth due to respiratory failure. Mice heterozygous for the Duttl/Robol deletion were bom at the expected frequency and had no obviously abnormal phenotype.
  • Duttl/Robol '1' mice failed to feed (Fig 3), were usually inactive with laboured breathing and nearly two-thirds (22/36) died within the first twenty-four hours.
  • Fig 3 At autopsy, lungs were frequently dark red (Fig. 3) and sank in fixative suggesting inadequate inflation. Following fixation and H&E staining, lungs from Duttl/Robol ' ' mice and their normal littermates were compared.
  • Example 9 Search for other phenotypic changes in Duttl/Robol ' ' ' mice. All newborn mice which die at birth were examined for macroscopic abnormalities in addition to their abnormal lungs. None was found except for three mice with diaphragmatic hernias. The heart, kidneys and muscle were examined microsopically but no abnormal histology was detected. In view of the function of Drosophila Robo in the control of axonal migration and the high level of expression of the protein in the brain of newborn mice (Fig. 2A) and in foetal brain (Clark, KJ, JX, EH and PHR unpublished observations), brains and spinal chords of mutant mice were examined.
  • Fig. 4F&H papillary hyperplasia was seen throughout the entire bronchial tree
  • Fig. 4E&G contrasting with the uniform cuboidal appearance of the bronchial epithelium of normal adult mice
  • focal dysplasia was observed characterised by increased epithelial cell layers, large pleomorphic nuclei and reduced cytoplasm (Fig. 41).
  • Fig. 41 focal dysplasia was observed characterised by increased epithelial cell layers, large pleomorphic nuclei and reduced cytoplasm.
  • Soluble Fab's were produced as described by Roovers et al (1998). The cultures were inoculated with E. coli TGI (K12, O(lac-pro), supE, thi, hsdD5/F' traD36, proA + B + , lacP, / ⁇ cZDM15) harbouring the Fab in pCESl . Individual bacterial clones were picked and production of soluble Fab was induced by activation of the upstream Lac Z promoter with isopropyl- ⁇ -1-thiogalactopyranoside (IPTG) as described by Marks et ⁇ / (1991).
  • IPTG isopropyl- ⁇ -1-thiogalactopyranoside
  • ELISA using soluble Fabs was performed on purified, recombinant DUTTl in order to identify binding Fabs from the individual clones selected.
  • ELISA plates (Costar, Cambridge, MA, USA) were coated overnight with 1 ⁇ g ml "1 DUTTl in phosphate-buffered saline (PBS), washed three times with PBS-T [PBS, 0.5% (v/v) Tween 20], three times with PBS and blocked for 1 h at room temperature (RT) with 2% MPBS [2% (w/v) Marvel - skimmed milk powder - in PBS].
  • induced bacterial supernatants were added [50% (v/v) in 2% MBS] and incubated for 1.5 h at RT. Bound antibody fragments were detected with a further antibody [50% (v/v) hybridoma supernatant in 2% MPBS], peroxidase-conjugated rabbit anti-mouse immunoglobins [Dako, Glustrup, Denmark; 0.1% (v/v) in 2% MPBS] and stained with trimethylbenzidine (TMB) and hydrogen peroxide. Optical density was measured at 450 nm. This assay is described in detail by Roovers et al (1998 ibid).
  • Competition ELISA For competition ELISA, DUTTl binding of soluble Fab antibody fragments was detected in the presence of excess whole murine monoclonal antibody.
  • the assay was carried out as described above but the wells were coated with antigens such as 10 ⁇ g/ml Bovine Serum Albumin (Sigma) in PBS, 3 mg/ml Hen Egg White Lysozyme (Boehringer Mannheim) in 0.1 M NaHCO 3 (pH 9.6), 10 ⁇ g/ml Tetanus Toxoid in 0.1 M NaHCO 3 (pH 9.6) and 0.5 ⁇ g/ml EGP-2 in PBS.
  • antigens such as 10 ⁇ g/ml Bovine Serum Albumin (Sigma) in PBS, 3 mg/ml Hen Egg White Lysozyme (Boehringer Mannheim) in 0.1 M NaHCO 3 (pH 9.6), 10 ⁇ g/ml Tetanus Toxoid in 0.1 M NaHCO 3 (pH 9.6) and 0.5 ⁇ g/ml EGP-2 in PBS.
  • antigens such as 10 ⁇ g/ml Bovine Serum Albumin (S
  • a polyclonal antisera was raised in rabbits against the C-terminal peptide of DUTTl/ROBOl (CYERGEDNNEELEETES) by Zeneca (Cambridge Research Biochemicals UK).
  • the antisera specifically recognised a band at approximately 190kDa in cells transfected with either a DUTTl or ROBO1 expression vector. Staining was blocked by preincubation of the immunising peptide with the antisera.
  • Tissues or whole embryos were fixed in 4% paraformaldehyde in PBS buffer for at least 24h, paraffin embedded and processed to give 4 micorometre sections. Sections were deparaffinised and endogenous peroxide quenched. After blocking, Duttl/Robol was detected using the polyclonal antisera described above followed by donkey biotin- conjugated anti-rabbit secondary antibody (SantaCruz USA) and amplification with the Vectastain ABC kit (Vector Laboratories USA) as per the manufacturers protocol. Positive staining was detected using nickel enhanced diaminobenzidine tetrahydrochloride (DAB) and counterstained with Fast Red. Slides were mounted with Vectashield-mounting medium. Staining was blocked by preincubation of the immunising peptide with the antisera. Sections were stained with haematoxylin and eosin (H&E).
  • H&E haematoxylin and eosin

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Abstract

La présente invention concerne des procédés destinés au diagnostic, à l'imagerie et au traitement de cellules cancéreuses. L'invention concerne plus particulièrement l'utilisation d'une molécule suppresseur de tumeur qui est apparente à des densités supérieures sur des cellules cancéreuses que sur des cellules se divisant normalement pour le diagnostic, l'imagerie, la prophylaxie et le traitement du cancer.
EP02765065A 2001-10-02 2002-10-02 Cancer Withdrawn EP1432821A2 (fr)

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US8940488B2 (en) 2004-03-31 2015-01-27 Hiroyuki Aburatani Cancer diagnosis and treatment of cancer using anti-robo 1 antibody
ATE476661T1 (de) * 2004-03-31 2010-08-15 Hiroyuki Aburatani Krebsdiagnose und behandlung unter verwendung eines anti-robo1-antikörpers
JP5117765B2 (ja) 2007-05-28 2013-01-16 国立大学法人 東京大学 抗ｒｏｂｏ１抗体を含むｐｅｔ用腫瘍診断剤
WO2010131590A1 (fr) * 2009-05-14 2010-11-18 国立大学法人 東京大学 Agent diagnostique et agent thérapeutique pour des tumeurs

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CA2306776A1 (fr) * 1997-11-14 1999-05-27 The Regents Of The University Of California Procedes permettant de moduler une fonction des cellules nerveuses

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