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EP1226172A1 - Compounds, antibodies and methods for treating, preventing or diagnosing tumour metastasis - Google Patents

Compounds, antibodies and methods for treating, preventing or diagnosing tumour metastasis

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Publication number
EP1226172A1
EP1226172A1 EP99960062A EP99960062A EP1226172A1 EP 1226172 A1 EP1226172 A1 EP 1226172A1 EP 99960062 A EP99960062 A EP 99960062A EP 99960062 A EP99960062 A EP 99960062A EP 1226172 A1 EP1226172 A1 EP 1226172A1
Authority
EP
European Patent Office
Prior art keywords
wnt
cancer
metastasis
cells
synthesised peptide
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Withdrawn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
EP99960062A
Other languages
German (de)
French (fr)
Inventor
Marzieh JÖNSSON
Tommy Experim. Pathology ANDERSSON
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Forskarpatent I SYD AB
Original Assignee
Forskarpatent I SYD AB
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Application filed by Forskarpatent I SYD AB filed Critical Forskarpatent I SYD AB
Publication of EP1226172A1 publication Critical patent/EP1226172A1/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • 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
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a novel synthesised peptide- analogue, including the functional domain of the nt-5a protein, a composition comprising the peptide-analogue.
  • the invention is also related to uses of the compound for the manufacture of a medicament or producing of a diagnostic agent in the treatment of metastasis or the diagnosing of metastasis. Furthermore, the invention relates to method of anti- metastasis treatment and diagnoses.
  • the metastasis process involves the entry of cells from the primary tumours into the blood and lymphatic circulation and their exit at metastatic sites.
  • Cells from different primary tumours do metastasise into different distal organs, for example breast tumours mainly metastasise into the bone marrow and the lungs.
  • breast tumours mainly metastasise into the bone marrow and the lungs.
  • About 70% of patients with advanced cancers suffer from distal metastases and thereby increased mortality.
  • Organ-conserving approaches are of high priority considering psychological devastation for the patients, although organ-preserving methods will increase the risk for cancer metastasis.
  • a phenomenon referred to as multiple drug resistance i.e. resistance to a wide range of structurally unrelated anticancer drugs, has caused several problems in treatment of various cancers with chemotherapeutic approaches. Even a patient receiving cytotoxic anti -cancer treatment is exposed to developing drug-resistant populations, which will multiply and eventually enter the circulation and infiltrate other organs. Thus, an anti-metastasis approach that functions in a wide variety of situations and tumour types, where a risk for the development of metastases exist, is of crucial significance .
  • tumour cells are a tumorogenic phenotype, which is transformed into a non-tumorogenic phenotype.
  • compositions and methods for transfection of tumour cells under conditions such that tumour growth is suppressed are transfected with the nucleic acid comprising the Wnt-5a gene.
  • the problem with this solution is that the tumour cells have to be transfected to suppress the tumour growth.
  • the present invention solves the problem by the use of synthesised peptide-analogue including the functional domain of the Wnt-5a protein in the surpressing or blocking of metastasis cells.
  • the advantage of using synthesised peptide-analogue including the functional domain of the Wnt-5a protein in therapeutic treatments is that it makes it possible for instance to avoid the use of virus particles.
  • the Wnt gene family encodes secreted signalling proteins, which are conserved in many species and are involved in em- bryogenesis. The signalling proteins are also involved in the regulation of a wide variety of normal and pathological proc- esses.
  • MMTV mouse mammary tumour virus
  • the discovery of a locus, which was activated in response to insertion of mouse mammary tumour virus (MMTV) led to identification of a mouse proto-oncogene called int-1 (for MMTV integration site) .
  • the int-1 gene was later shown to be homologous to the Drosophila wingless (wg) gene, which is involved in embryonic segmentation and the combination of these two terms gave rise to the name 'WNT'.
  • wg Drosophila wingless
  • the signals derived from extracellular Wnt proteins are transmitted and sensed by neighbouring cells through an auto- crine or paracrine mechanism.
  • the diversity of function between different members of the Wnt family allows them to be divided into two distinct classes. These classes are designated as the Wnt-1 and the Wnt-5a class, which are described by Dale, T. C. Biochem. J. (1998) 329 : 209-223.
  • genes of the Wnt-1 class are capable of cell transformation and tumourigenesis .
  • the Wnt-1 and IVnt-3 genes of the Wnt-1 class can induce tumour growth in mouse mammary tissue when they are activated by insertion of mouse mammary tumour virus.
  • the Wnt -5a expression was found to induce adhesion of mammary epithelial cells to collagen, which is a dominant extracellular component associated with the periductal matrix in breast tissue. This induced ability to bind tightly onto the matrix was accompanied by phosphorylation and activation of discoidin domain 1 tyrosine kinase receptors, which was described by Vogel, W. FASEB. Supp. (1999) p. 77 - 82. In addition, cells expressing Wnt-5a protein proved to resist the inductive effect of hepatocyte growth factor (Gherardi, E. et al . Cancer Cells . (1991) 3:227-232) on cell motility, whereas Wnt-5a antisense cells invade the surrounding matrix extensively.
  • the treatment with the synthesised peptide-analogue which includes the functional domain of the Wnt-5a protein according to the present invention, will prevent the entry of disseminated cancer cells into blood or lymphatic circulation.
  • Various methods can be applied for supplying the compound (s) to the tumour cells under such conditions that cancer metastasis is blocked.
  • the present invention also contemplates the use of diagnostic methods for prediction of risk factor for the development of invasive carcinoma and recurrence. It has surprisingly been found that the Wnt-5a protein strengthens cell to matrix interaction. Therefore, it is predictable that a peptide-analogue comprising the functional domain of the Wnt-5a protein, can generate signals that force the disseminated cells to adhere effectively to the matrix. Accordingly, free movement of the cells and thereby ability to metastasise will be blocked.
  • the Wnt-5a protein sequence (published sequence accessible in several gene banks)
  • one embodiment of the present invention is a synthesised peptide-analogue, which includes the functional domain of the Wnt-5a protein. This domain is encoded from at least a part of the Wnt -5a gene for the treatment or the pre- vention of tumour metastasis.
  • Another embodiment of the present invention is a composition including a synthesised peptide-analogue.
  • Another embodiment of the present invention is an antibody derived from immunisation in mammals with the synthesised peptide-analogue or the composition.
  • the antibody is used as a diagnostic agent in diagnosing metastasis derived from all types of solid tumours such as breast cancer, prostatic can- cer, colon cancer or melanoma cancer.
  • Another embodiment of the present invention is the use of the synthesised peptide-analogue for the production of a medicament for the inhibiting of disseminated cells free movement and thereby blocking the metastasis .
  • Another embodiment of the present invention is a method of treatment or prevention of metastasis derived from all types of solid tumours such as breast cancer, prostatic cancer, colon cancer or melanoma cancer etc.
  • a further embodiment of the present invention is to provide a method for diagnosing cancer, comprising use of Wnt-5a antibody provided by invention.
  • invasive tumours can easily be detected when cancer disease is suspected.
  • This diagnostic step will provide guidance for radiation and chemo-therapeutic treatments.
  • Preferred embodiments and other aspects of the present invention are defined in the dependent claims.
  • the present invention thus provides a new composition com- prising the functional domain of tumour suppressor protein Wnt-5a, under conditions that prevent tumour metastasis.
  • the invention contemplates application of the Wnt-5a peptide with a variety of treatment methods .
  • the interval of rest periods can be extended to increase life quality of the patient with- out taking any risk for tumour cell dissemination and recurrence.
  • This method will eliminate the need for radiation for patients with negative lymph node metastasis and patient with positive lymph node will benefit from decreased radiation dose or intensity in their therapy.
  • the application can ra- tionally be combined with chemo-therapeutic regimens without recourse to other potentially confounding treatments.
  • expression of Wnt-5a is restricted to epithelial and stem cells, the Wnt-5a peptide will not interfere with the movement of other motile cells such as periphery blood cells.
  • the invention provides among others access to and insight in strengthening of cell to matrix adhesion by initiation of Wnt-5a signalling pathway in cells other than mammary epithelial as well.
  • Figure 1 Expression of Wnt-5a normalised the morphology of MCF-7 breast cancer cells.
  • A Morphology of MCF-7 untrans- fected parental cells.
  • B Morphology of MCF-7 control cells.
  • C Morphology of the MCF-7 cells expressing Wnt-5a protein.
  • Figure 2 The cell to collagen binding ability of various HB2 cell populations. The values represent the average number of cells attached to collagen during 1-hour incubation. The assay was performed in triplicate wells coated with various concentrations of collagen I as indicated.
  • Figure 3 Wnt-5a antisense cells invade the surrounding collagen matrix. The micrographs show: (A) The structures formed by control cells. (B) Structures formed by Wnt-5a-expressing cells that were primarily ball-like. (C) Structures generated by Wnt-5a antisense cells were mostly cyst-shaped. (D) To quantify the results, 100 morphogenetic structures were examined. Each of the structures were examined on triplicate plates and classified as cyst shaped, branching, or ball- like; the mean values of the number of structures in each class were then calculated and plotted in the illustrated diagram. Bar, 80 ⁇ m.
  • FIG. 4 Wnt-5a antisense cells show an increased capacity to invade matrix in response to the HGF stimulation.
  • the mi- crographs show: (A) Structures produced by control cells.
  • D The illustrated diagram was created by quantifying the morphogenetic structures as described in fig. 8. Bar, 80 ⁇ m.
  • FIG. 5 Example of Wnt-5a lost in invasive breast tumours.
  • the ductal carcinoma cells infilterating surrounding tissue have lost expression (single arrowhead) of Wnt-5a protein while the cells found in ductal in situ express this protein strongly (double arrowheads) .
  • the C-terminal end of the Wnt -5a gene was tagged with a HA (hemagglutinin) epitope and cloned into plasmid Bluescript KS+ (Shimizu, H. et al . Cell Growth Differ. (1997) 8 : 1349- 58) .
  • the Wnt-5a-HA cDNA was removed from pBluescript and sub- cloned into a retroviral vector, pLNCX (Miller, A.D. et al . Bio . Tech . (1989) 7:980-990).
  • neomycin phosphotransferase expression is controlled by the murine leukemia virus long terminal repeat
  • Wnt-5a-HA cDNA transcription is controlled by an internal CMV enhancer/promoter.
  • a full-length Wnt -5a cDNA was subcloned in antisense orientation (3 ' -5' ) .
  • HB2 human normal breast epithelial cell line
  • Various popula- tions of HB2 cells expressing the pLNCX empty vector (control) , the pLNCX-Wnt-5a-HA, or the pLNCX-Wnt-5a-HA in antisense orientation were isolated by growing the stable transfectants in the presence of 500 ⁇ g/ml geneticin. These cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10 ⁇ g/ml bovine insulin, and 5 ⁇ g/ml hydrocortisone . All cell types were incubated at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide .
  • MCF-7 breast cancer cells were transfected with Wnt-5a cDNA and isolated stable transfectants expressing Wnt-5a-HA (Wnt-5a-expressing) and empty vector (control) under similar conditions as described for HB2 transfectants .
  • a Wnt-5a polyclonal antiserum was generated in rabbits immunised with a synthesised peptide corresponding to the C- terminal region of human and mouse Wnt-5a protein.
  • the serum was purified in two steps:
  • the anti-Wnt-5a antibody was used to screen several cell lines for expression of the Wnt-5a protein. The obtained results from cell lines for detection of the Wnt-5a protein were consistent with previous reports of Wnt-5a expression at the messenger RNA levels. Also isolated Wnt-5a antisense transfectants proved to express varying amounts of Wnt -5a protein, providing additional evidence of the specificity of the Wnt -5a antibody.
  • the experimental cells including Wnt-5a-overexpressing, antisense and control cells were detached from the tissue cul- ture plates using cell scrapers, and single-cell suspensions were prepared and washed twice in serum-free medium. Aliquots of single-cell suspensions (1 ml) containing 5 x 10 3 cells were plated on 24 well plates, coated with solutions of neutralised bovine dermal collagen type I (Vitrogen-100 , Colla- gen Corp.) in concentrations ranging from 1 to 100 ⁇ g/ml. The cells were incubated for 2 hours at 37°C. The wells were then gently washed three times with phosphate-buffered saline (PBS) to remove non-adherent cells.
  • PBS phosphate-buffered saline
  • Attached cells were fixed with 1% glutaraldehyde and stained with crystal violet (0.1% in H 2 0) , and the wells were subsequently washed with PBS three times. The relative number of attached cells in each well was evaluated by measuring absorbance at 540 nm in an ELISA Microplate Reader. The mean values (triplicate wells) for each concentration and population were calculated, and from those values, we subtracted non-specific cell adhesion measured on BSA-coated wells. As shown in Figure 2, the collagen-binding capacity of cells was significantly impaired in Wnt-5a antisense cells, as compared to Wnt-5a-overexpressing and control cells demonstrating a positive correlation between Wnt-5a protein expression and cell to matrix adhesion.
  • Single-cell suspensions of the experimental cell populations were prepared. Aliquots of the cell suspension were mixed with a neutralised collagen type I solution at a 1 : 10 ratio to give a final concentration of 1 x 10 5 cells/ml. The cell- collagen mixtures were then added to tissue culture plates (6 cm in diameter) and allowed to polymerise at 37°C for 1 hour.
  • the cells were either incubated in standard growth medium (Figure 3) or in standard growth medium supplemented with recombinant human HGF ( Figure 4) .
  • the cells were allowed to grow within the collagen gel for 7-8 days.
  • the appearance of produced morphogenetic structures was documented with a Nikon F 301 camera connected to a Nikon TMS inverted microscope.
  • the growth medium, with or without HGF, was replaced with fresh medium every other day.
  • the cells were either incubated in standard growth medium (Figure 3) or in standard growth medium supplemented with recombinant human HGF ( Figure 4) .
  • the cells were allowed to grow within the collagen gel for 7-8 days.
  • the appearance of produced morphogenetic structures was documented with a Nikon F 301 camera connected to a Nikon TMS inverted microscope.
  • the growth medium, with or without HGF, was replaced with fresh medium every other day.
  • the number of each type of structure was expressed as a percentage of the total number of structures present in the gel and the mean values were subsequently calculated.
  • the number of the generated cyst -shaped and branching structures by antisense cells was approximately 2 -fold compared to that produced by Wnt-5a-overexpressing or control cells.
  • Figure 4 shows a comparison of the cell branching in- tensity induced by HGF (an indicator of cell motility and invasion) in experimental cell populations.
  • the number of branches produced by antisense cells proved to be greater than 2-fold compared to that of control cells and 6-fold compared to that produced by Wnt-5a-overexpressing cells.
  • the rabbit polyclonal anti-Wnt-5a antibody was diluted 1:2000 and used under similar conditions as described above.
  • samples of the lysates were pre- cleared with protein-A Sepharose alone and subsequently incubated with 3 ⁇ g of anti-DDRl (Santa Cruz Biotechnology) or anti-phosphotyrosine antibody (4G10, Upstate Biotechnology, Inc.).
  • antigen-antibody complexes were collected using protein-A Sepharose.
  • the proteins were then separated on 8% SDS-polyacrylamide gels under reducing conditions and electrophoretically trans- ferred onto nitrocellulose membranes (Bio-Rad Laboratories) .
  • the membranes were incubated with anti-DDRl or 4G10 antibody.
  • Antigen-antibody association was detected using horseradish peroxidase conjugated secondary antibodies (DAKO, Denmark) and an enhanced chemiluminescence (ECL) detection system.
  • the DDR1 receptor was found to be expressed at a similar level in Wnt-5a-overexpressing, control, and antisense cells, but that suppression of the Wnt-5a protein led to significantly lower tyrosine phosphorylation of these receptors in the Wnt-5a antisense cells.
  • DDR1 protein was expressed in equal level in control, Wnt-5a-expressing, and parental MCF-7 cells. However, only the Wnt-5a-expressing cells showed a significant elevation of tyrosine phosphorylation of the DDR1 receptors.
  • Immuno-histochemical analysis of tumours were prepared on 3 - 5 ⁇ m sections cut on poly-L-lysine coated slides. Antigen retrieval was performed by incubating the dewaxed sections in a temperature-controlled microwave oven. The sections were allowed to cool to room temperature and were blocked with 1% BSA-PBS for non-specific antibody binding. Sections were then incubated with an anti-Wnt-5a antibody overnight at 4°C. A standard alkaline phosphatase technique was used for visualisation, and the expression was evaluated with a Leitz Diaplan microscope using 10 and 25x magnifying objectives.

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Abstract

The present invention relates to a novel synthesised peptide-analogue, including the functional domain of the Wnt-5a protein, a composition comprising the synthesised peptide-analogue, an antibody used as a diagnostic agent. The present invention also relates to uses of the compound for the manufacture of a medicament or a diagnostic agent in the treatment of metastasis or the diagnosing of metastasis.

Description

COMPOUNDS, ANTIBODIES AND METHODS FOR TREATING, PREVENTING OR DIAGNOSING TUMOUR METASTASIS .
The present invention relates to a novel synthesised peptide- analogue, including the functional domain of the nt-5a protein, a composition comprising the peptide-analogue. The invention is also related to uses of the compound for the manufacture of a medicament or producing of a diagnostic agent in the treatment of metastasis or the diagnosing of metastasis. Furthermore, the invention relates to method of anti- metastasis treatment and diagnoses.
The metastasis process involves the entry of cells from the primary tumours into the blood and lymphatic circulation and their exit at metastatic sites. Cells from different primary tumours do metastasise into different distal organs, for example breast tumours mainly metastasise into the bone marrow and the lungs. About 70% of patients with advanced cancers suffer from distal metastases and thereby increased mortality.
Although effective treatment is available for early-stage cancers, there is an obvious lack of effective therapies for metastatic diseases. These remain incurable for the majority of patients, despite extensive clinical evaluation of multi- pie chemotherapeutic or hormonal regimens. A balance between a good life quality for the patient and an efficient treatment is of high priority in clinical trial when designing a treatment protocol for patients. However, there still remain a number of situations to consider as risks for metastasis for example:
A) Organ-conserving approaches are of high priority considering psychological devastation for the patients, although organ-preserving methods will increase the risk for cancer metastasis.
B) In addition to the fact that the recurrence of the various cancers, when invasive, carries with it an increased risk of death, the potential cost for extensive chemotherapy or the necessity of additional surgery has to be considered.
C) A phenomenon referred to as multiple drug resistance, i.e. resistance to a wide range of structurally unrelated anticancer drugs, has caused several problems in treatment of various cancers with chemotherapeutic approaches. Even a patient receiving cytotoxic anti -cancer treatment is exposed to developing drug-resistant populations, which will multiply and eventually enter the circulation and infiltrate other organs. Thus, an anti-metastasis approach that functions in a wide variety of situations and tumour types, where a risk for the development of metastases exist, is of crucial significance .
D) During the treatment of cancer, the entire treatment protocol extends over several months, during which various combinations of drugs are given, interspersed with several rest periods. The latter intervals are necessary in order to allow the host to recover from the toxic effects of the various chemicals given. However, during these rest periods the neoplastic population will expand and infiltrate other organs eventually.
To solve these problems an invention is disclosed in WO
98/23730, where the use of antisense Wnt-5a as well as expressing of the Wnt-5a gene product in tumour cells is contemplated. Such a tumour cell is a tumorogenic phenotype, which is transformed into a non-tumorogenic phenotype. For the treatment of cancer and metastasis without radiation or toxic chemoterapeutic agents compositions and methods for transfection of tumour cells under conditions such that tumour growth is suppressed. The cells are transfected with the nucleic acid comprising the Wnt-5a gene.
The problem with this solution is that the tumour cells have to be transfected to suppress the tumour growth. The present invention solves the problem by the use of synthesised peptide-analogue including the functional domain of the Wnt-5a protein in the surpressing or blocking of metastasis cells. The advantage of using synthesised peptide-analogue including the functional domain of the Wnt-5a protein in therapeutic treatments is that it makes it possible for instance to avoid the use of virus particles.
Background of the nt-gene
The Wnt gene family encodes secreted signalling proteins, which are conserved in many species and are involved in em- bryogenesis. The signalling proteins are also involved in the regulation of a wide variety of normal and pathological proc- esses. The discovery of a locus, which was activated in response to insertion of mouse mammary tumour virus (MMTV) , led to identification of a mouse proto-oncogene called int-1 (for MMTV integration site) . The int-1 gene was later shown to be homologous to the Drosophila wingless (wg) gene, which is involved in embryonic segmentation and the combination of these two terms gave rise to the name 'WNT'.
The signals derived from extracellular Wnt proteins are transmitted and sensed by neighbouring cells through an auto- crine or paracrine mechanism. The diversity of function between different members of the Wnt family allows them to be divided into two distinct classes. These classes are designated as the Wnt-1 and the Wnt-5a class, which are described by Dale, T. C. Biochem. J. (1998) 329 : 209-223.
As regards genes of the Wnt-1 class, they are capable of cell transformation and tumourigenesis . For example it has been shown that the Wnt-1 and IVnt-3 genes of the Wnt-1 class can induce tumour growth in mouse mammary tissue when they are activated by insertion of mouse mammary tumour virus.
However, genes of the Wnt-5a class, including Wnt -5a itself, are non-transforming and can interestingly antagonise the effects of transforming Wnts through unknown mechanisms. Torres, M. A. et al . has studied these effects. The results are published in J. Cell Biol . (1996) 133:1123-1137.
It has been shown that inhibition of the Wnt-5a messenger RNA mimics the Wnt-1 -mediated cell transformation, this is described by Olson, D. J. et al . in the article: Antisense Wnt- 5a mimics Wnt-1-mediated C57mg mammary epithelial cell transformation. Exp . Cell Res . (1998) 241:134-141. Thus, the Wnt- 5a protein seems to be capable of promoting signals that counteract the oncogenic pathways of components activated during cell malignancy.
The Wnt -5a expression was found to induce adhesion of mammary epithelial cells to collagen, which is a dominant extracellular component associated with the periductal matrix in breast tissue. This induced ability to bind tightly onto the matrix was accompanied by phosphorylation and activation of discoidin domain 1 tyrosine kinase receptors, which was described by Vogel, W. FASEB. Supp. (1999) p. 77 - 82. In addition, cells expressing Wnt-5a protein proved to resist the inductive effect of hepatocyte growth factor (Gherardi, E. et al . Cancer Cells . (1991) 3:227-232) on cell motility, whereas Wnt-5a antisense cells invade the surrounding matrix extensively.
Detailed description of the invention
To study mechanisms, by which Wnt-5a could inhibit tumour growth and cell transformation, an antisense technique was applied to suppress endogenous expression of Wnt-5a protein in a human normal breast cell line. Wnt-5a suppression in normal cells was found to induce an elongated cell phenotype resembling that of transformed cells. Interestingly, expression of Wnt-5a in a breast cancer cell line, MCF-7, that has lost Wnt-5a expression, could normalise the morphology of the cancer cells. The effect on morphology of cancer cells was associated with a compact and contact inhibited cell phenotype at confluence. Thus, these demonstrations support a role for the human Wnt-5a gene in regulation of the morphology of mammary cells.
To analyse how Wnt-5a expression was associated with other clinical variables 110 primary breast tumours were examined. The variables such as tumour staging, size, expression of oestrogen and progesterone, lymph node metastasis or age of patient etc. were analysed, the tumours were divided into three subgroups, namely ductal (DCA) , lobular carcinomas (LCA) and ductal cancer in situ (DCIS) subgroup for expression of Wnt-5a protein. The results show that Wnt-5a expression is lost or weakly expressed in stage III tumours. The abnormality of Wnt- 5a expression is also associated with tumour size and oestrogen (ER) and progesterone (PgR) expression.
Thus, the results strongly demonstrate that the Wnt-5a protein participates in regulation of signals running from the extracellular matrix through the DDR1 receptor and thereby provides a correct interaction between matrix components and cell surface receptors. This property appears to be relevant in controlling the motility and invasiveness of the cells during tumourigenesis, and loss of this expression contributes to tumour progression.
The treatment with the synthesised peptide-analogue, which includes the functional domain of the Wnt-5a protein according to the present invention, will prevent the entry of disseminated cancer cells into blood or lymphatic circulation. Various methods can be applied for supplying the compound (s) to the tumour cells under such conditions that cancer metastasis is blocked.
A prospective demonstration in predicting the behaviour of a number of non-invasive carcinoma, such as ductal carcinoma in situ (DCIS) or fibrocystic disease, is currently lacking. The present invention also contemplates the use of diagnostic methods for prediction of risk factor for the development of invasive carcinoma and recurrence. It has surprisingly been found that the Wnt-5a protein strengthens cell to matrix interaction. Therefore, it is predictable that a peptide-analogue comprising the functional domain of the Wnt-5a protein, can generate signals that force the disseminated cells to adhere effectively to the matrix. Accordingly, free movement of the cells and thereby ability to metastasise will be blocked.
As regards clinical evaluation of invasive tumours, a pro- spective demonstration in predicting risk for tumour recurrence is currently lacking. As the intensity of cancer treatment is tailored to an individual risk for recurrence in the clinic, quantification of the levels of Wnt-5a expression using an anti-Wnt-5a antibody in tumours, will highlight pa- tients with high risk for recurrence, thus providing guidance in the selection of treatment or radiation approaches. As loss of Wnt-5a expression is highly restricted to invasive and low differentiated tumours, the quantification of Wnt-5a expression will be informative in determination of clinical staging or differentiation.
The Wnt-5a protein sequence (published sequence accessible in several gene banks)
1 magsamsskf flvalaiffs faqwieans wwslgmnnpv qmsevyiiga qplcsqlagl
61 sqgqkklchl yqdhmqyige gaktgikecq yqfrhrrwnc stvdntsvfg rvmqigsret
121 aftyavsaag wnamsracr egelstcgcs raarpkdlpr dwlwggcgdn idygyrfake 181 fvdarereri hakgsyesar ilmnlhnnea grrtvynlad vackchgvsg scslktcwlq
241 ladfrkvgda lkekvdsaaa mrlnsrgklv αvnsrfnspt tqdlv idps pdycyrnest
301 gslgtqgrlc nktsegmdgc elmccgrgyd σfktvqterc hckfhwccyv kckkcteivd
361 σfvck The C-terminal region of Wnt-5a protein (underlined) is regarded to be the region of the presence of functional do- main(s). Therefore, this sequence serves as a suitable sequence for synthesising of peptide-analogue (s) .
Accordingly, one embodiment of the present invention is a synthesised peptide-analogue, which includes the functional domain of the Wnt-5a protein. This domain is encoded from at least a part of the Wnt -5a gene for the treatment or the pre- vention of tumour metastasis.
Another embodiment of the present invention is a composition including a synthesised peptide-analogue.
Another embodiment of the present invention is an antibody derived from immunisation in mammals with the synthesised peptide-analogue or the composition. The antibody is used as a diagnostic agent in diagnosing metastasis derived from all types of solid tumours such as breast cancer, prostatic can- cer, colon cancer or melanoma cancer.
Another embodiment of the present invention is the use of the synthesised peptide-analogue for the production of a medicament for the inhibiting of disseminated cells free movement and thereby blocking the metastasis .
Another embodiment of the present invention is a method of treatment or prevention of metastasis derived from all types of solid tumours such as breast cancer, prostatic cancer, colon cancer or melanoma cancer etc.
A further embodiment of the present invention is to provide a method for diagnosing cancer, comprising use of Wnt-5a antibody provided by invention. For example, invasive tumours can easily be detected when cancer disease is suspected. This diagnostic step will provide guidance for radiation and chemo-therapeutic treatments. Preferred embodiments and other aspects of the present invention are defined in the dependent claims.
The present invention thus provides a new composition com- prising the functional domain of tumour suppressor protein Wnt-5a, under conditions that prevent tumour metastasis. The invention contemplates application of the Wnt-5a peptide with a variety of treatment methods . The interval of rest periods can be extended to increase life quality of the patient with- out taking any risk for tumour cell dissemination and recurrence. This method will eliminate the need for radiation for patients with negative lymph node metastasis and patient with positive lymph node will benefit from decreased radiation dose or intensity in their therapy. The application can ra- tionally be combined with chemo-therapeutic regimens without recourse to other potentially confounding treatments. As expression of Wnt-5a is restricted to epithelial and stem cells, the Wnt-5a peptide will not interfere with the movement of other motile cells such as periphery blood cells.
The invention provides among others access to and insight in strengthening of cell to matrix adhesion by initiation of Wnt-5a signalling pathway in cells other than mammary epithelial as well.
The present invention will be further described in more detail in the following examples, tables and figures.
Figure 1: Expression of Wnt-5a normalised the morphology of MCF-7 breast cancer cells. (A) Morphology of MCF-7 untrans- fected parental cells. (B) Morphology of MCF-7 control cells. (C) ; Morphology of the MCF-7 cells expressing Wnt-5a protein.
Figure 2: The cell to collagen binding ability of various HB2 cell populations. The values represent the average number of cells attached to collagen during 1-hour incubation. The assay was performed in triplicate wells coated with various concentrations of collagen I as indicated. Figure 3: Wnt-5a antisense cells invade the surrounding collagen matrix. The micrographs show: (A) The structures formed by control cells. (B) Structures formed by Wnt-5a-expressing cells that were primarily ball-like. (C) Structures generated by Wnt-5a antisense cells were mostly cyst-shaped. (D) To quantify the results, 100 morphogenetic structures were examined. Each of the structures were examined on triplicate plates and classified as cyst shaped, branching, or ball- like; the mean values of the number of structures in each class were then calculated and plotted in the illustrated diagram. Bar, 80 μm.
Figure 4: Wnt-5a antisense cells show an increased capacity to invade matrix in response to the HGF stimulation. The mi- crographs show: (A) Structures produced by control cells. (B) Structures produced by Wnt-5a-overexpressing cells that were primarily ball-like, with only limited branching and cyst formation. (C) Structures produced by Wnt-5a antisense cells showing an extensive generation of branching and cyst-shaped structures. (D) The illustrated diagram was created by quantifying the morphogenetic structures as described in fig. 8. Bar, 80 μm.
Figure 5: Example of Wnt-5a lost in invasive breast tumours. The ductal carcinoma cells infilterating surrounding tissue have lost expression (single arrowhead) of Wnt-5a protein while the cells found in ductal in situ express this protein strongly (double arrowheads) .
Example 1: Cloning of Wnt-5a
The C-terminal end of the Wnt -5a gene was tagged with a HA (hemagglutinin) epitope and cloned into plasmid Bluescript KS+ (Shimizu, H. et al . Cell Growth Differ. (1997) 8 : 1349- 58) . The Wnt-5a-HA cDNA was removed from pBluescript and sub- cloned into a retroviral vector, pLNCX (Miller, A.D. et al . Bio . Tech . (1989) 7:980-990). In the pLNCX vector, neomycin phosphotransferase expression is controlled by the murine leukemia virus long terminal repeat, and Wnt-5a-HA cDNA transcription is controlled by an internal CMV enhancer/promoter. For antisense experiments, a full-length Wnt -5a cDNA was subcloned in antisense orientation (3 ' -5' ) .
Thereafter, a human normal breast epithelial cell line, HB2 , was transfected with the described vectors. Various popula- tions of HB2 cells expressing the pLNCX empty vector (control) , the pLNCX-Wnt-5a-HA, or the pLNCX-Wnt-5a-HA in antisense orientation were isolated by growing the stable transfectants in the presence of 500 μg/ml geneticin. These cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 10 μg/ml bovine insulin, and 5 μg/ml hydrocortisone . All cell types were incubated at 37°C in a humidified atmosphere of 95% air and 5% carbon dioxide .
Furthermore, the MCF-7 breast cancer cells were transfected with Wnt-5a cDNA and isolated stable transfectants expressing Wnt-5a-HA (Wnt-5a-expressing) and empty vector (control) under similar conditions as described for HB2 transfectants .
In the experiments, etopic expression of the Wnt-5a protein did not alter the morphology of normal breast epithelial cells, whereas suppression of endogenous Wnt-5a protein in antisense cells resulted in an appearance resembling that of transformed cells. In line with this, expression of the Wnt- 5a protein in MCF-7 cells normalised the morphology of the cancer cells providing a compact and differentiated phenotype (Figure 1) . Thus, the results support a role for Wnt-5a in maintenance of normal morphology of mammary epithelial cells.
EXAMPLE 2: Generation of the Wnt-5a antibody
A Wnt-5a polyclonal antiserum was generated in rabbits immunised with a synthesised peptide corresponding to the C- terminal region of human and mouse Wnt-5a protein. The serum was purified in two steps:
1) all IgGs in the antiserum were collected using a protein-A Sepharose column;
2) the eluted fraction was further purified on a Sepharose
4B column (Amersham Pharmacia Biotech) containing the immobilised Wnt-5a peptide used for immunisation. The affinity- purified antibody was then eluted with 0.1 M glycine.
The anti-Wnt-5a antibody was used to screen several cell lines for expression of the Wnt-5a protein. The obtained results from cell lines for detection of the Wnt-5a protein were consistent with previous reports of Wnt-5a expression at the messenger RNA levels. Also isolated Wnt-5a antisense transfectants proved to express varying amounts of Wnt -5a protein, providing additional evidence of the specificity of the Wnt -5a antibody.
EXAMPLE 3 : Cell to matrix binding assay
The experimental cells including Wnt-5a-overexpressing, antisense and control cells were detached from the tissue cul- ture plates using cell scrapers, and single-cell suspensions were prepared and washed twice in serum-free medium. Aliquots of single-cell suspensions (1 ml) containing 5 x 103 cells were plated on 24 well plates, coated with solutions of neutralised bovine dermal collagen type I (Vitrogen-100 , Colla- gen Corp.) in concentrations ranging from 1 to 100 μg/ml. The cells were incubated for 2 hours at 37°C. The wells were then gently washed three times with phosphate-buffered saline (PBS) to remove non-adherent cells. Attached cells were fixed with 1% glutaraldehyde and stained with crystal violet (0.1% in H20) , and the wells were subsequently washed with PBS three times. The relative number of attached cells in each well was evaluated by measuring absorbance at 540 nm in an ELISA Microplate Reader. The mean values (triplicate wells) for each concentration and population were calculated, and from those values, we subtracted non-specific cell adhesion measured on BSA-coated wells. As shown in Figure 2, the collagen-binding capacity of cells was significantly impaired in Wnt-5a antisense cells, as compared to Wnt-5a-overexpressing and control cells demonstrating a positive correlation between Wnt-5a protein expression and cell to matrix adhesion.
EXAMPLE 4: Cell to matrix invasion assay
Single-cell suspensions of the experimental cell populations were prepared. Aliquots of the cell suspension were mixed with a neutralised collagen type I solution at a 1 : 10 ratio to give a final concentration of 1 x 105 cells/ml. The cell- collagen mixtures were then added to tissue culture plates (6 cm in diameter) and allowed to polymerise at 37°C for 1 hour.
The cells were either incubated in standard growth medium (Figure 3) or in standard growth medium supplemented with recombinant human HGF (Figure 4) . The cells were allowed to grow within the collagen gel for 7-8 days. The appearance of produced morphogenetic structures was documented with a Nikon F 301 camera connected to a Nikon TMS inverted microscope. The growth medium, with or without HGF, was replaced with fresh medium every other day.
The cells were either incubated in standard growth medium (Figure 3) or in standard growth medium supplemented with recombinant human HGF (Figure 4) . The cells were allowed to grow within the collagen gel for 7-8 days. The appearance of produced morphogenetic structures was documented with a Nikon F 301 camera connected to a Nikon TMS inverted microscope. The growth medium, with or without HGF, was replaced with fresh medium every other day.
The number of each type of structure was expressed as a percentage of the total number of structures present in the gel and the mean values were subsequently calculated. As shown in Figure 3, the number of the generated cyst -shaped and branching structures by antisense cells was approximately 2 -fold compared to that produced by Wnt-5a-overexpressing or control cells. Figure 4 shows a comparison of the cell branching in- tensity induced by HGF (an indicator of cell motility and invasion) in experimental cell populations. The number of branches produced by antisense cells proved to be greater than 2-fold compared to that of control cells and 6-fold compared to that produced by Wnt-5a-overexpressing cells. Thus, the results demonstrate that suppression of Wnt-5a facilitates penetration of cells through the surrounding matrix, and this effect is amplified in response to the inductive effects of HGF.
EXAMPLE 5: Immuno-blots and immuno-precipitations
To detect Wnt-5a-HA fusion protein, 50 μg of total protein was separated on 12% SDS-polyacrylamide gel. The proteins were then electrophoretically transferred onto nitrocellulose membranes, which were incubated with an anti-HA antibody
(Berkeley Antibody Co., CA) for 1 hour at room temperature. To detect the endogenous Wnt-5a protein levels in the cells, the rabbit polyclonal anti-Wnt-5a antibody was diluted 1:2000 and used under similar conditions as described above.
For immunoprecipitation, samples of the lysates were pre- cleared with protein-A Sepharose alone and subsequently incubated with 3 μg of anti-DDRl (Santa Cruz Biotechnology) or anti-phosphotyrosine antibody (4G10, Upstate Biotechnology, Inc.). The antigen-antibody complexes were collected using protein-A Sepharose.
The proteins were then separated on 8% SDS-polyacrylamide gels under reducing conditions and electrophoretically trans- ferred onto nitrocellulose membranes (Bio-Rad Laboratories) . The membranes were incubated with anti-DDRl or 4G10 antibody. Antigen-antibody association was detected using horseradish peroxidase conjugated secondary antibodies (DAKO, Denmark) and an enhanced chemiluminescence (ECL) detection system.
The DDR1 receptor was found to be expressed at a similar level in Wnt-5a-overexpressing, control, and antisense cells, but that suppression of the Wnt-5a protein led to significantly lower tyrosine phosphorylation of these receptors in the Wnt-5a antisense cells. Interestingly DDR1 protein was expressed in equal level in control, Wnt-5a-expressing, and parental MCF-7 cells. However, only the Wnt-5a-expressing cells showed a significant elevation of tyrosine phosphorylation of the DDR1 receptors.
EXAMPLE 6: Immuno-histochemistry of tumours
Immuno-histochemical analysis of tumours were prepared on 3 - 5 μm sections cut on poly-L-lysine coated slides. Antigen retrieval was performed by incubating the dewaxed sections in a temperature-controlled microwave oven. The sections were allowed to cool to room temperature and were blocked with 1% BSA-PBS for non-specific antibody binding. Sections were then incubated with an anti-Wnt-5a antibody overnight at 4°C. A standard alkaline phosphatase technique was used for visualisation, and the expression was evaluated with a Leitz Diaplan microscope using 10 and 25x magnifying objectives.
Statistical analysis supported this findings showing strong inverse correlation between Wnt-5a protein levels and the tumour stage (P = 0.006) . This means that Wnt-5a expression was strongly associated with the degree of differentiation, i.e. low differentiated tumours express Wnt-5a very weakly or they have lost this expression while early stage tumours, in situ and high differentiated tumours have retained this expression (Figure 5) . EXAMPLE 7: Relation of Wnt-5a protein levels with axillary lymph node metastases
To further confirm that the loss is associated with cell me- tastasis we examined levels of Wnt-5a protein in lymph node invading epithelial cells, stained on an average 5 lymph nodes positive for cell metastases from each patient . The overall rate of metastasis to auxiliary lymph nodes in patients with invasive ductal and lobular carcinoma proved to be greater than 90%. Thus, these data together with the data above strongly support that Wnt -5a expression has indeed an anti-metastasis effect on tumour cells and loss of this expression is a prediction for cell invasion and metastasis.
Above Examples are intended to illustrate certain preferred embodiments and aspects of the present invention, and are not constructed to limit the scope of the claimed invention.

Claims

1. A synthesised peptide-analogue, including the functional domain of the Wnt-5a protein, which is encoded from at least a part of the Wnt-5a gene for the treatment or the prevention of tumour metastasis.
2. The synthesised peptide-analogue according to claim 1, wherein the synthesised peptide is encoded from the entire Wnt-5a gene.
3. The synthesised peptide-analogue according to claim 1, wherein the synthesised peptide is encoded from the 241- position of the Wnt-5a gene.
4. The synthesised peptide-analogue according to claim 1, wherein the synthesised peptide is encoded from the 301- position of the Wnt-5a gene.
5. The synthesised peptide-analogue according to any of claims 1 - 4, wherein the tumour metastasis is derived from all types of solid tumours such as breast cancer, prostatic cancer, colon cancer, melanoma cancer etc.
6. A composition, comprising the synthesised peptide- analogue according to any of claims 1 - 5, for the treatment or the prevention of tumour metastasis.
7. An antibody derived from immunisation in mammals with the synthesised peptide-analogue according to any of claims 1
- 5 or the composition according to claim 6 for use as a diagnostic agent in diagnosing metastasis.
8. An antibody according to claim 7, characterised in that the metastasis is derived from all types of solid tumours such as breast cancer, prostatic cancer, colon cancer or melanoma cancer etc.
9. A diagnostic agent, comprising the synthesised peptide-analogue according to any of claims 1 - 5 or the composition according to claim 6 for use as a diagnostic agent in diagnosing metastasis.
10. Use of the synthesised peptide-analogue according any of claims 1 - 5, for the production of a pharmaceutical composition for the inhibiting of disseminated cells free movement and thereby blocking the metastasis.
11. Use of the synthesised peptide-analogue according any of claims 1 - 5, for the production of a medicament for the treatment of tumour metastasis derived from all types of solid tumours such as breast cancer, prostatic cancer, colon cancer melanoma cancer etc.
12. Use of the synthesised peptide-analogue according any of claims 1 - 5, for the production of a diagnostic agent for the diagnosing of cancer.
13. A method of treatment or prevention of metastasis derived from all types of solid tumours such as breast cancer, prostatic cancer, colon cancer, melanoma cancer etc., in a human patient suffering therefrom which method comprises administration to said patient an effective amount of the synthesised peptide-analogue defined in any of claims 1 - 5.
14. A method of diagnosing of metastasis derived from all types of solid tumours such as breast cancer, prostatic can- cer, colon cancer or melanoma cancer etc., in a human patient suffering therefrom which method comprises administration to said patient an effective amount of the synthesised peptide- analogue defined in any of claims 1 - 5.
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