WO2011161217A2 - Targeting of vegfr2 - Google Patents

Targeting of vegfr2 Download PDF

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WO2011161217A2
WO2011161217A2 PCT/EP2011/060563 EP2011060563W WO2011161217A2 WO 2011161217 A2 WO2011161217 A2 WO 2011161217A2 EP 2011060563 W EP2011060563 W EP 2011060563W WO 2011161217 A2 WO2011161217 A2 WO 2011161217A2
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vegfr2
cells
antagonist
tumour
subject
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PCT/EP2011/060563
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French (fr)
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WO2011161217A3 (en
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Petra Hamerlik
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Palacký University in Olomouc
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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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2863—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for growth factors, growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies

Definitions

  • the invention relates to methods of identifying and killing cancer stem-like cells using compounds capable of associating with VEGFR2. Furthermore, the invention relates to identification of compounds useful for identifying and killing cancer stem-like cells and/or progenitor cells, said compounds being VEGFR2 antagonists. The present invention does also relate to identification of patients that are sensitive to VEGFR2 antagonist and hence would benefit from treatment according to the present invention.
  • Malignant astrocytomas the most common subtype of primary brain tumours, are aggressive, highly invasive, and neurologically destructive tumours considered being among the deadliest of human cancers (Maher EA, Furnari, FB, Bachoo RM, Rowitch DH, Louis DN, Cavenee WK, DePinho R. Malignant glioma: genetics and biology of grave matter. Genes & Development. 2001 ; 15: 131 1 -1332.).
  • Standard chemotherapy approaches have only resulted in modest improvements in time to progression and survival for patients with brain tumours (reviewed in Newton HB. Molecular neuro-oncology and development of targeted therapeutic strategies for brain tumours.
  • VEGF is the key factor involved in angiogenesis. Beside its pro-angiogenic function, VEGF also increases vascular permeability and was found to be over-expressed in a variety of tumours including highly vascularized and infiltrative astrocytomas (grade lll/IV), where VEGF expression correlates with tumour progression and poor prognosis. Interactions between tumour cells and their microenvironment are critically important in the biology of cancer, and include growth factor signalling in paracrine and autocrine manners.
  • Angiogenesis a process highly regulated by a large number of pro- and anti- angiogenic factors, is essential not only in early embryogenesis but also in tumour development and progression. Angiogenesis also takes place during tissue growth and repair; female reproductive cycle, foetal development and inflammation. A wide range of pro- and anti-angiogenic factors are deregulated in brain tumours and released after a variety of physiological and pathological stimuli. Localized breakdown of extracellular matrix (ECM) precedes the proliferation, migration and tissue infiltration of endothelial cells. In time these cells remodel back into capillary structures, and a new ECM is deposited.
  • ECM extracellular matrix
  • VEGF vascular endothelial growth factor
  • VEGF has been highlighted as a multi-functional chemokine stimulating differentiation, survival, proliferation, migration, tubulogenesis and vascular permeability in endothelial cells. It is required for normal development of blood vessels, and targeted inactivation of only a single allele of the VEGF during embryogenesis in mice is lethal. There is a strong relationship between VEGF expression and tumour aggressiveness, metastatic potential, a short time to relapse and consequently, elevated VEGF expression indicates poor prognosis in patients with cancer.
  • Angiogenesis is one of the most critical factors in tumour maintenance and progression. Malignant cells become capable of inducing phenotypic changes in endothelial cells as well as other cell types. Highly malignant astrocytomas may exhibit striking angiogenesis and markedly increased expression of VEGF. VEGF exerts multiple effects at the cellular level as a result of its ability to affect a complex integrated network of signalling pathways via three cognate receptors on surface of endothelial cells: VEGFR1 (Flt-1 ), VEGFR2 (KDR) and VEGFR3, and two non-tyrosine neuropilin receptors: NRP-1 and NRP-2.
  • VEGFR1 Flt-1
  • VEGFR2 VEGFR2
  • NRP-1 and NRP-2 two non-tyrosine neuropilin receptors
  • NRP-1 and NRP-2 are isoform-specific receptors of VEGF, which function as co-receptors with VEGFR2.
  • a soluble truncated form of VEGFR1 (sFlt-1 ) binds VEGF as strongly as the full-length Flt-1 .
  • sFlt-1 inhibits VEGF activity by sequestering it from signalling receptors and by forming non- signalling heterodimers with VEGFR2 (the so called decoy receptor).
  • the VEGF receptors are primarily expressed in tumour vascular endothelium, and are absent in normal endothelial cells in surrounding tissue (Plate KH, Breier G, Weich HA, Mennel HD, Risau W. Vascular endothelial growth factor and glioma angiogenesis: coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms. Int J Cancer. 1994; 59:520-529).
  • the VEGFR2 antagonist Tivozanib (available from Aveo Pharmaceuticals Inc.) has demonstrated the overall median progression-free survival of patients in phase 2 trials to be 1 1 .8 months.
  • the present invention demonstrates how targeting of VEGFR2 either directly or indirectly increases the sensitivity of cancer stem-like cells and/or progenitor cells to conventional cancer therapy and an increase in progression-free survival time is expected compared to conventional cancer therapy alone.
  • the present invention demonstrates that VEGFR2 is expressed in cancer stem-like cells and progenitor cells and is required for their survival.
  • VEGFR2 By targeting VEGFR2 directly with antagonists to VEGFR2 or indirectly with antagonists to NRP-1 either alone or in combination, thereby inhibiting the activity of VEGFR2, it is possible to make the cancer stem-like cells and/or progenitor cells more susceptible to cancer therapy including radiation therapy.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a vascular endothelial growth factor receptor 2 (VEGFR2) antagonist for treatment of a tumour selected from the group consisting of connective tissue tumours, tumours of the nervous system, hematologic tumours, lymphoid leukemias and/or myeloid leukemias in a subject in need thereof, wherein the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy.
  • VEGFR2 vascular endothelial growth factor receptor 2
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a neuropilin-1 (NRP-1 ) antagonist for treatment of a tumour selected from the group consisting of connective tissue tumours, tumours of the nervous system, hematologic tumours, lymphoid leukemias and/or myeloid leukemias in a subject in need thereof, wherein the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy.
  • NPP-1 neuropilin-1
  • the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a vascular endothelial growth factor receptor 2 (VEGFR2) antagonist for reducing the risk of recurrence of a VVEGFR2 (VEGFR2) antagonist for reducing the risk of recurrence of a VVEGFR2 (VEGFR2) antagonist for reducing the risk of recurrence of a VVEGFR2 (VEGFR2) antagonist for reducing the risk of recurrence of a VVEGFR2 (VEGFR2) antagonist for reducing the risk of recurrence of a
  • VAGFR2 vascular endothelial growth factor receptor 2
  • the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy. In another embodiment, the pharmaceutical composition is prepared for administration to said subject prior to radiation therapy which would follow within days or weeks of administering said pharmaceutical composition.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising a therapeutically effective amount of a neuropilin-1 (NRP-1 ) antagonist for reducing the risk of recurrence of a hyperproliferative disease in a subject in need thereof by inhibiting growth of cancer stem-like cells.
  • the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy.
  • the present invention enables identification of cancer stem-like cells and/or progenitor cells expressing VEGFR2 by identifying cells expressing VEGFR2. According, it is also an objective of the present invention to identify and optionally isolate cancer stem-like cells and/or progenitor cells. In one embodiment the identification of cancer stem-like cells and/or progenitor cells expressing VEGFR2 can be used for evaluating the presence and putative progression of a cancer disease. In one embodiment the present invention relates to a method for isolation of cancer stem-like cells and/or progenitor cells expressing VEGFR2 comprising contacting the population of cells with a molecule that specifically binds VEGFR2, and isolating cells that bind the molecule that specifically binds VEGFR2.
  • the present invention relates to a method of treating a tumour in a subject by inhibiting the cancer stem-like cells and/or progenitor cells expressing VEGFR2 by administering to the subject a therapeutically effective amount of a vascular endothelial growth factor receptor 2 (VEGFR2) antagonist and/or an antagonist of NRP-1 , and immediately thereafter subjecting the tumour in the subject to ionizing radiation therapy and optionally treatment with anti-VEGF antibodies.
  • the method also relates to reducing the risk of recurrence of a tumour in a subject by inhibiting the cancer stem-like cells and/or progenitor cells.
  • the VEGFR2 is operatively linked to a reporter gene and the activation of said reporter gene is measured.
  • yet another aspect of the present invention provides the use of the compound as identified herein for the manufacture of a medicament for the treatment of a cancer disease.
  • the medicament is for reducing the risk of recurrence of a cancer disease.
  • FIG. 1 VEGFR2 was found to be expressed in astroglial cells on human glioblastoma specimens, associated with the so-called "vascular niche". Expression of VEGFR2 co-localizes with expression of the commonly used cancer stem-like cell marker - CD133. Presence of VEGFR2 positive cells was further confirmed by FACS in 6 other human specimens and the percentage of positive cells ranged from 0.8-35% (data not shown), where there was always presence of double-positive CD133/VEGFR2 cells.
  • Figure 2 Co-staining with CD31 was done to exclude that the VEGFR2 positive glial cells are actually glioblastoma-derived tumour endothelial cells. Staining on frozen section from human specimen was performed. Co-staining of VEGFR2 with CD31 was done to determine the presence of tumour endothelial cell in our target fraction. As shown above, there is definitely fraction of VEGFR2 positive and CD31 negative cells.
  • FIG. 3 Tumour sphere formation assay was performed on 3 different specimens. This assay is used to study self-renewing capacity (property of stem-like cells) of cells. We sorted one cell per well and were counting number of spheres (of more then 50 cells) formed by single cell. As shown above, CD133-VEGFR2 positive and single VEGFR2 positive show high tumour sphere forming capacity, compared to single CD133 positive and negative population.
  • FIG. 4 Growth properties of VEGFR2 positive cells are significantly enhanced compared to their negative counterparts.
  • FIG. 5 Shows unspecific binding of the two VEGFR2 selective inhibitors used at different concentrations, all below the concentration expected to target other tyrosine kinase receptors like EGFR, PDGFR. Unspecific binding in the present invention is binding of an inhibitor which is not specific to the VEGFR2 exclusively. In this figure two known VEGFR2 inhibitors is demonstrated to also bind VEGFR2 negative cells, hence the two inhibitors must have other targets and are not specific to VEGFR2.
  • FIG. 6 IR-induced CD133-positive cancer stem-like cells' enrichment is abrogated by SU1498 in vitro.
  • Astroglial tumour cells A172 and U251 MG were plated at 60% density.
  • cells were treated with ionizing radiation of 8 Gy (dose rate 2.18 Gy/min by an X-ray generator, Pantak, Berkshire, United Kingdom; HF160; 150 kV; 15mA) after 1 hour pre-treatment with the inhibitor SU1498 or left untreated, and analyzed at indicated time after irradiation for the presence of CD133-positive cells using FACS Calibur (BD Biosciences).
  • Figure 7 A). Specimen #1966 did not show statistically significant difference in in vivo tumour formation assay when 100 cells injected.
  • the label High denotes high level of VEGFR2, and the label Low denotes low level of VEGFR2.
  • FIG. 8 Radiation does not efficiently kill VEGFR2 positive cells alone.
  • Cells were freshly dissociated (from mouse xenograft #556 (A and B) and from patient #0607 (C and D)), recovered over night, plated and treated for 2 hours with the VEGFR2 antagonist SU1498 or left untreated. After this the cells were irradiated with 8 Gy or left without radiation. 24 hours (A and C) and 72 hours later (B and D), surviving cells were analyzed by FACS for presence of CD133, VEGFR2 and VEGFR2-CD133 positive cells.
  • VEGFR2+ means small sub-population of cells which attach and resembles endothelia cells.
  • VEGFR2 expression in gliomas VEGFR2 is expressed by human glioma initiating cells (GICs).
  • GICs human glioma initiating cells
  • glioma cells not only express membrane VEGFR2, but a substantial fraction ranging from 12 to 27% cells have VEGFR2 internalized as measured by FACS analysis of extracellular versus total VEGFR2 staining (C) and can be detected directly by immunohistochemistry staining on paraffin-embedded sections from corresponding glioma specimens ( * labels membrane localization, ⁇ labels internalized VEGFR2) (B).
  • VEGFR2-positive cells recapitulate patient phenotype when injected in immunocompromised rodent host.
  • VEGFR2 is autophosphorylated (B), which indicates constitutive activation of VEGFR2 receptor in these cells.
  • B autophosphorylated
  • VEGFR2 expression in gliomas accounts for growth advantage and increased VEGF secretion
  • VEGFR2-low (L; surface negative), VEGFR2-medium (M; surface negative) and VEGFR2-high (H; surface positive) cells were sorted out to confirm the presence of internalized VEGFR2 by Western blot analysis (see on the right).
  • VEGFR2-low cells do not have internalized VEGFR2, whereas it could be detected in VEGFR2-medium cells.
  • neuropilinl (NRP1 ) protein levels were gradually increased with increased VEGFR2 expression indicating importance of neuropilins in VEGFR2 signaling.
  • VEGFR2- high cells have significantly high self-renewing capacities in sphere formation assay and enhanced viability in vitro (C).
  • VEGFR2-high cells exhibit enhanced secretion of VEGF ligand when compared to VEGFR2-negative cells (D).
  • Most common antiangiogenic drug AVAstin (bevacizumab) efficiently binds and blocks VEGF ligand as expected and SU1498 tyrosine kinase inhibitor (VEGFR2 specific), decreases VEGF secretion indication of autocrine loop signaling (E).
  • Fig.12 VEGFR2 is stabilized by NRP1 and remains constitutively active in endosomal compartment
  • Freshly dissociated xenografted #556 cells were left to recover overnight and next day treated as follows: 10 mM SU1498 (A) or Bevacizumab (B, 0.5 mg/ml) pre-treated or not treated for 2 hours and further irradiated or sham-irradiated (8 Gy).
  • Cells were collected 12 and 24 hours post-IR treatment and analyzed using FACS for VEGFR2-AlexaFluor647/AnnexinV-AlexaFluor488/7-AAD co-staining. For analysis, cells were sub-gated using FlowJo software as VEGFR2 high and VEGFR2
  • each sub-population was separately analyzed for the presence of apoptotic (AnnexinV/7-AAD co-staining) and viable cell fractions.
  • VEGFR2 tyrosine kinase inhibitor SU1498 (alone, or followed by IR) significantly inhibited cell growth followed over 7-day period (C). This effect was lacking when Bevacizumab was used.
  • Fig.14 Lentiviral mediated shRNA targeting of VEGFR2 results in enhanced apoptosis, decreased viability and impaired tumor formation
  • Kaplan-Meier survival curves demonstrate decreased survival when cells high in VEGFR2 expression are transplanted into the right frontal lobe of immunocompromised mice (p ⁇ 0.01 for 100 #556 cells) tested with log-rank analysis of survival curves. Injection of 1000, 5000 or 10000 cells did not show any growth advantages and increase in tumor formation of VEGFR2 high cells.
  • VEGFR2 knockdown led to increase in cell death as evaluated by relative caspase 3/7 activity compared to non-targeting shRNA (shNT). Increased induction of apoptosis was accompanied by decreased cell viability over a period of 7 days (D) as assessed by CellTiter Glo Kit (Promega).
  • Fig.15 VEGFR2 represents a protein with very short half-life and is induced by ionizing radiation.
  • VEGFR2 high cells (#556) were sorted using FACS sorting, left recover overnight in GF free medium and then treated with cyclohexamide for 30 min., 1 hour, 3 hours, 6 hours or left untreated and analyzed for VEGFR2 protein stability using semiquantitative Western blot analysis. Photoshop software was employed to calculate raw and relative intensities of corresponding bands. Further, the half-life of 42 minutes was estimated. Graph with exponential decay curve used for half-life estimation is shown (y- axis represents normalized relative intensity; x-axis represents Time in minutes). (B) MG132 (proteosomal inhibitor) was used in VEGFR2 high cells to investigate further protein stabilization/accumulation after proteasomal inhibition.
  • VEGFR2 protein levels indicating of strong impact of protesomal degration pathway on VEGFR2 protein turnover.
  • C To analyze to effect of ionizing radiation (IR) on VEGFR2-protein level, we treated VEGFR2- expressing cells with the dose of 8 Gy of IR and used Western blot analysis to check for total and phosphorylated VEGFR2 levels. As shown here, radiation induces total and phospho-VEGFR2 expression and pro-survival VEGFR2 signaling over 48 hours post radiation. At 72 hours pos radiation, VEGFR2 levels returned to baseline expression a compared to control. Detailed description of the invention
  • treating refers to reversing, alleviating, inhibiting the process of, the disease, disorder or condition to which such term applies, or one or more symptoms of such disease, disorder or condition and includes the administration of an antagonist to alleviate the symptoms or the complications, or eliminate the disease, condition, or disorder.
  • treatment is curative or ameliorating.
  • the antagonist can be administered to prevent the disease or the onset of the symptoms or the complications of the disease.
  • anti-neoplastic compound or "anti-neoplastic agent” as used herein, unless otherwise indicated, denotes a cytotoxic compound which can be used as cancer chemotherapy to inhibit and combat cancer cells and tumours.
  • Anti-neoplastic compound or “anti-neoplastic agent” can also be used to denote immune stimulating agents, antibodies, and cancer vaccines as described in the section “combination treatment” herein below.
  • proliferative disease or “hyperproliferative disease” as used herein, unless otherwise indicated, denotes a disease in which tissue, such as cells, grow or spread at a rapid abnormal rate, such as for example in cancer.
  • terapéuticaally effective amount denotes an amount of a molecule which exerts a relevant therapeutically effect in a subject to which the molecule is administered.
  • immediate prior to radiation therapy denotes an administration within 5 days prior to initiation of radiation therapy, more preferably within 4 days, such as within 3 days, more preferably within 2 days, such as within 1 day, more preferably within 20 hours, such as within 10 hours, more preferably within 5 hours, more preferably within 4.5 hours, such as within 4 hours, more preferably within 3.5 hours, such as within 3 hours, more preferably within 2.5 hours, such as within 2 hours, more preferably within 1 .5 hours, such as within 1 hour, more preferably within 0.5 hour, such as within 15 minutes, more preferably within 10 minutes, such as within 5 minutes prior to radiation therapy.
  • before radiation therapy denotes an administration within 3 weeks prior to initiation of radiation therapy, more preferably with 2 weeks, such as within 1 week, more preferably within 6 days.
  • before treatment with anti-VEGF antibodies denotes an administration within 5 days prior to administration of anti-VEGF antibodies, more preferably within 4 days, such as within 3 days, more preferably within 2 days, such as within 1 day prior to administration of anti-VEGF antibodies.
  • Biological activity refers to the biologically useful effects of a molecule on a specific cell.
  • a biologically active compound is one which when administered to a subject exerts its effect on a target cell.
  • radiationosensitizing drug as used herein, unless otherwise indicated, denotes a drug which increases the sensitivity of targeted tumour cells towards radiotherapy.
  • subject denotes a living animal.
  • the subject is a mammal, including humans and non-human mammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats and mice.
  • the subject is a human.
  • reducing the risk of recurrence denotes a reduced risk of recurrent cancer as described in the section "recurrent cancer", so that at the most 90% of the subjects demonstrates a recurrent cancer, more preferably at the most 80%, such as at the most 70%, more preferably at the most 60%, such as at the most 50%, more preferably at the most 40%, such as at the most 30%, more preferably at the most 20%, such as at the most 10%, more preferably at the most 5%, such as at the most 4%, more preferably at the most 3%, such as at the most 2%, most preferably at the most 1 %.
  • the method of the present invention is especially useful for reducing the risk of recurrence if the VEGFR2 antagonist of the present invention is combined with radiation therapy as described in the section "radiation therapy”
  • cancer stem-like cell denotes cells with proliferative potential, said cells expressing a variety of different markers, such as CD133, VEGFR2, and integrin Alpha-6.
  • progenitor cell denotes a biological cell, which has a tendency to differentiate into a specific cell type. The progenitor cell is more specific than a stem cell and may be directed to differentiate into its "target” cell. The most important difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, whereas progenitor cells can only divide a limited number of times.
  • recurrence and "recurrent hyperproliferative disease” as used herein, unless otherwise indicated, denotes that a hyperproliferative disease which has been subject to treatment is reoccurring in the same patient.
  • a recurrent hyperproliferative disease is a cancer, which has come back in a patient who was thought to be cancer-free or in remission after treatment.
  • the hyperproliferative disease such as cancer can come back:
  • distant recurrence Distant recurrence is also called metastatic recurrence.
  • the hyperproliferative disease might recur in distant parts of body, such as in bones, the liver, or the lungs. This may happen because some cancer cells, in particular cancer stem-like cells, have broken off from the original tumour, travelled through the body, and are establishing tumours elsewhere.
  • a cancer When the patient is evaluated to be free of tumours using conventional detection methods employed in the field of oncology, a cancer is considered to be in remission (the temporary or permanent absence of disease).
  • a recurrence is defined as the return of cancer after a period of remission.
  • cancer recurs because undetected surviving cancer cells in particular cancer stem-like cells frequently remain in the body after treatment. Over time, these undetected cells can multiply and grow large enough to be recognized and diagnosed using the conventional methods of diagnosis employed in the field of oncology today. Depending on the type of cancer, this can happen in weeks, months, or even many years after the primary (original) cancer was treated.
  • the likelihood that a cancer will come back depends on the type of the primary cancer. When and where a cancer will recur also varies. Most cancers have a predictable pattern of recurrence. Brain tumours and especially astrocytoma is a cancer type with a high rate of recurrence. In one embodiment of the present invention the risk of recurrence, as defined above, is reduced.
  • the present invention relates to targeting cancer stem-like cells and/or progenitor cells expressing VEGFR2. Furthermore the present invention describes antagonists of VEGFR2. In particular the invention describes methods for eliminating cancer stem-like cells by inhibiting VEGFR2 signalling through antagonist blocking of the receptor rendering the cancer stem-like cells and/or progenitor cells sensitive to conventional cancer treatment such as radiation therapy.
  • the inhibition of VEGFR2 signalling may be directly or indirectly. In preferred embodiment the inhibition is however direct. Indirect inhibition of VEGFR2 signalling is preferably through NRP-1 receptor. Inhibition of VEGFR2 may in one embodiment be accomplished by a combination of VEGFR2 inhibitors and NRP-1 inhibitors.
  • the invention describes methods for detecting cancer stem-like cells and/or progenitor cells by identifying the presence of VEGFR2 expressing cells in a cell sample, said cell sample preferably obtained from a patient suffering from a hyperproliferative disease.
  • the detection of cancer stem-like cells and/or progenitor cells is in one embodiment performed by identifying the presence of a variety of other markers, such as CD133 preferably in combination with the identification of the presence of VEGFR2.
  • the presence of VEGFR2 may in one embodiment be identified using antibodies directed against VEGFR2, such as antibodies describes in the section "Antagonists"
  • Cancer stem-like cells and/or progenitor cells are cells with proliferative potential, said cells expressing a variety of different markers, such as CD133, VEGFR2, and integrin Alpha-6. Cancer stem-like cells and/or progenitor cells expressing a high level of VEGFR2 demonstrates the capability of tumour formation when only 100 cells are injected in a subject (example 1 and figure 7).
  • CSCs cancer stem-like cells
  • FACS fluorescence activated cell sorting
  • AC133 human CD133 antigen
  • cancer stem-like cells Besides expressing the CD133 antigen, cancer stem-like cells express mRNAs for several additional recognized NSC markers, including bmi-1 , Sox2 and musashi-1 (Hemmati HD, Nakano I, Lazareff JA, Masterman-Smith M, Geschwind DH, Bronner-Fraser M, Kornblum HI. Cancerous stem-like cells can arise from pediatric brain tumors. Proc Natl Acad Sci U S A. 2003; 100: 15178-83). Some cancer stem-like cells have been found to CD133-negative, and hence CD133 is not necessarily the best marker of cancer stem-like cells. The present invention demonstrated that VEGFR2 is expressed also in CD133-negative cancer stem-like cells.
  • Cancer stem-like cells may be cultivated in vitro e.g. brain tumour-derived cancer stem- like cells (BTCSCs) may be cultured according to the following criteria: i) Expression of NSC markers CD133 and nestin (a marker of glial cells); ii) Generation of spheres morphologically indistinguishable from neurospheres; iii) Self renewal and proliferation; and iv) Production of differentiated progeny in vitro or recapitulation of the parental tumour mass growth when implanted into immunodeficient animals.
  • BTCSCs brain tumour-derived cancer stem- like cells
  • CD133-positive human cells were sufficient to form brain tumours as xenografts in NOD-SCID mice (Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB. Identification of human brain tumour initiating cells. Nature. 2004; 432: 396-401 ).
  • the CD133-positive BTCSCs also exhibit high resistance to current cytotoxic drugs and radiotherapy, in contrast to their CD133-negative counterparts (Liu G, Yuan X, Zeng Z, Tunici P, Ng H, Abdulkadir IR, Lu L, Irvin D, Black KL, Yu JS.
  • VEGFR2 is enriched in cancer stem-like cells in brain tumours.
  • VEGFR2 positive cells demonstrate self-renewing capacity (property of stem-like cells) of cells.
  • CD133- VEGFR2 positive and single VEGFR2 positive cells show high tumour sphere forming capacity (a measurement of self-renewing capacity), compared to single CD133 positive and negative population.
  • the present invention demonstrates that by targeting VEGFR2 expressing cancer stem-like cells, said cancer stem-like cells being CD133-positive, the sensitivity of the cancer stem-like cells to conventional cancer treatment is increased.
  • the conventional cancer treatment is radiotherapy.
  • the present invention relates to VEGFR2 antagonists, said antagonist described in the section "antagonists".
  • the antagonist of the present invention may be used for inhibiting the signalling through the wild-type VEGFR2 (SEQ ID NO:1 ).
  • the VEGRF2 is involved in angiogenesis, differentiation and host-virus interactions.
  • VEGFR2 is also denoted KDR/Flk-1 and is a 152 kDa single-pass type 1 membrane protein. It belongs to the protein kinase superfamily and the Tyr protein kinase family.
  • VEGFR2 contains seven Ig-like (Immunoglobulin-like) C2-type domains located in the extracellular part and one protein kinase domain located in the intracellular part.
  • the protein kinase domain is located on amino acid 834-1 162 of SEQ ID NO:1 .
  • the present invention also relates to antagonists of functional homologues of VEGFR2.
  • functional homologues is VEGFR2 variants with the same function as wildtype VEGFR2, said functional homologous having changes in the amino acid sequence as compared to SEQ ID NO:1 .
  • Common to all functional homologous of the present invention is the conservation of Arg82, Lys84 and His86 of VEGFR2 (numbering according to SEQ ID NO:1 ).
  • the present invention relates to functional homologues of VEGFR2 antagonists.
  • a functional homologue is a polypeptide that exhibits some sequence identity with a known VEGFR2 antagonist as the antagonist described in the section "antagonists", sharing at least 50%, preferably at least 60%, more preferably at least 75%, even more preferably at least 80%, yet more preferably at least 85%, even more preferably at least 90%, yet even more preferably at least 95%, and most preferably at least 99% identity with known VEGFR2 antagonists.
  • Sequence identity can be calculated using a number of well-known algorithms and applying a number of different gap penalties. In relation to the present invention the sequence identity should be calculated relative to the full-length sequence of a known VEGFR2 antagonist. Any sequence alignment tool, such as but not limited to FASTA, BLAST, or LALIGN may be used for searching homologues and calculating sequence identity. Moreover, when appropriate any commonly known substitution matrix, such as but not limited to PAM, BLOSSUM or PSSM matrices, may be applied with the search algorithm. VEGFR2 is almost exclusively expressed in endothelial cells, however the present invention has revealed that VEGFR2 also is present in cancer stem-like cells and/or progenitor cells.
  • VEGFR2 in cancer stem-like cells and/or progenitor cells has been found to be present in both CD133 negative cancer cells as well as CD133 positive cancer stem-like cells.
  • Cancer stem-like cells and/or progenitor cells expressing a high level of VEGFR2 demonstrate the capability of tumour formation when only 100 cells are injected in a subject (example 1 and figure 7).
  • the present invention demonstrates that by targeting VEGFR2 expressing cancer stem-like cells and/or progenitor cells, the sensitivity of the cancer stem-like cells and/or progenitor cells to conventional cancer treatment is increased.
  • the conventional cancer treatment is radiotherapy.
  • the present invention demonstrates that by targeting VEGFR2 with a VEGFR2 antagonist prior to radiation the effects of radiation-induced VEGF secretion is minimised and the efficiency of the radiation treatment hence increased by targeting the unique features of the candidate cancer stem-like cells and/or progenitor cells.
  • a combined strategy may include available or future VEGFR2 antagonists or antibodies to inhibit VEGFR2 as described in the section "antagonist", combined with standard DNA damaging treatment modalities such as ionization radiation therapy and alkylating drugs, complemented by selective inhibitors of checkpoint signalling or DNA repair to counteract the mechanisms underlying tumour resistance to such treatments.
  • the combined strategy for treatment may be made as multiple treatments during longer periods of time.
  • the present invention relates to a method of detecting a cancer stem-like cell and/or progenitor cells expressing VEGFR2 in a population of isolated cells, said cells preferably being isolated from a tumour.
  • cancer stem-like cells and “VEGFR2” cancer stem-like cells and/or progenitor cells expressing VEGFR2 are important for the treatment resistance of tumours to conventional cancer therapy.
  • Cancer stem-like cells and/or progenitor cells of the present invention holds self renewal potential and proliferative potential and said cancer stem-like cells and/or progenitor cells expresses VEGFR2, preferably without expression of CD31 , CD31 being a marker for endothelial cells.
  • the present invention demonstrates that by targeting VEGFR2 expressing cancer stem-like cells and/or progenitor cells, said cancer stem-like cells and/or progenitor cells being CD133- positive, the sensitivity of the cancer stem-like cells and/or progenitor cells to conventional cancer treatment is increased.
  • the conventional cancer treatment is radiotherapy.
  • the conventional cancer treatment is administration of anti-VEGF antibodies.
  • Said tumour is in one embodiment selected from the group consisting of connective tissue tumours, tumours of the nervous system, hematologic tumours, lymphoid leukemias and/or myeloid leukemias.
  • tumour is a tumour of the nervous system, more preferably a tumour selected from the group consisting of glioma, glioblastoma, glioblastoma multiforme, astrocytoma, oligodendroglioma, ependymomas, medulloblastoma, meningioma, neurinoma and Schwannoma, even more preferably the tumour is a glioblastoma or glioblastoma multiforme.
  • the steps of the method according to the invention could in one embodiment be performed under conditions suitable for maintaining the cell morphology i.e. the cytoplasm and nucleus of the cells.
  • the invention relates to a method of detecting a cancer stem-like cell and/or progenitor cells expressing VEGFR2 in a population of isolated cells comprising the following steps:
  • the molecule that specifically binds VEGFR2 is labelled by a detectable label.
  • the label may be any of the labels described herein below in the section "Labelling".
  • the molecule that specifically binds VEGFR2 may also be detected using another molecule that specifically binds said molecule that specifically binds VEGFR2, wherein said other molecule is labelled using a detectable label.
  • the detectable label it may be detected using conventional methods. If the detectable label is a fluorescent label it may be detected using e.g. a fluorescence microscope, plate reader or a FACS.
  • distinguishing said endothelial cells may be done by contacting said sample with a molecule specifically binding endothelial cells.
  • Said molecule specifically binding endothelial cells may in a preferred embodiment be labelled by a detectable label, more preferably different detectable label from the label of the molecule that specifically binds VEGFR2.
  • the molecule that specifically binds endothelial cells may be detected by another molecule specifically binding thereto.
  • Said other molecule may then be labelled by a detectable label.
  • the sample may then be subjected to a separation step, e.g. by FACS analysis.
  • the sample is a tissue section mounted on a glass slide and distinguishing said endothelial cells may be done by evaluating the tissue section contacted with said molecules, said molecules preferably being labelled, for the presence of the specific molecules.
  • the molecule that specifically binds VEGFR2 is an antagonist as described in the section "antagonist" and the molecule that specifically binds endothelial cells is an antibody selected from the group consisting of antibodies specifically binding one or more selected from the group consisting of CD31 , CD34, Tusc5, 7B4, BNH9/BNF13, CD105, CD146, D2-40, EN4 and PAL-E.
  • said antibody specifically binds CD31 .
  • the endothelial cells may be removed from the sample by contacting said sample with a molecule specifically binding endothelial cells, said molecule such as an antibody specific for the endothelial cells.
  • Said molecule, such as an antibody may preferably be coupled to a solid support e.g. a column or another suitable material.
  • said antibody is selected from the group consisting of antibodies specifically binding CD31 , CD34, Tusc5, 7B4, BNH9/BNF13, CD105, CD146, D2-40, EN4 and PAL-E.
  • said antibody specifically binds CD31 . Any cells not binding the specific molecule can be removed by simple washing and subjected to further analysis according to the present invention.
  • the cancer stem-like cell and/or progenitor cells expressing VEGFR2 may be detected using various suitable techniques.
  • the cancer stem-like cells and/or progenitor cells expressing VEGFR2 are selectively labelled.
  • the selective labelling of the cancer stem-like cells and/or progenitor cells expressing VEGFR2 is done by the specific recognition of the cancer stem-like cells and/or progenitor cells expressing VEGFR2 by a specific molecule, said molecule preferably being an antibody, said antibody preferably being a VEGFR2 specific antibody.
  • detection of cancer stem-like cells and/or progenitor cells expressing VEGFR2 is performed by co-labelling the cell sample with a molecule that specifically binds VEGFR2, wherein said molecule may be any of the VEGFR2 antagonists being described in the section "antagonists", and one or more other cancer stem-like cell and/or progenitor cells expressing VEGFR2 marker.
  • detection of cancer stem-like cells and/or progenitor cells expressing VEGFR2 is performed by co-labelling the cell sample with VEGFR2 specific molecule and CD133 and/or integrin Alpha-6.
  • the detection of cancer stem-like cells expressing VEGFR2 is performed by co-labelling the cell sample with VEGFR2 specific molecule and CD133.
  • detection of cancer stem-like cells and/or progenitor cells expressing VEGFR2 is performed so as to detect both membrane expressed VEGFR2 and internalized VEGFR2. This is done by dual labelling. Firstly, dissociated tumour cells (e.g. paraffin sections) are subjected to primary-conjugated VEGFR2 antibody which would detect the membrane expressed VEGFR2. After washing out non-bound antibody, cells are fixed, permeabilized and co-labelled with the same antibody to achieve the total staining for VEGFR2.
  • VEGFR2 Only a small fraction of total VEGFR2 is present on the surface of cancer stem-like cells and/or progenitor cells as demonstrated in figure 9.
  • detection of cancer stem-like cells and/or progenitor cells expressing VEGFR2 may in one embodiment be used for diagnosis of patients suffering from a hyperproliferative disease, preferably a cancer.
  • a hyperproliferative disease preferably a cancer.
  • the amount of VEGFR2 expressing cancer stem-like cells and/or progenitor cells expressing VEGFR2 could in one embodiment be used as an indication of the prognosis for said hyperproliferative disease in said subject.
  • the detection of a high number of detected cancer stem-like cells and/or progenitor cells expressing VEGFR2 increases the severity of the hyperproliferative disease and the prognosis for the subject is hence not good.
  • the detection of cancer stem-like cells and/or progenitor cells expressing VEGFR2 may be used to identify patients sensitive to treatment with VEGFR2 and/or NRP-1 antagonist as described herein below in the section "Method of treatment”.
  • the detection of cancer stem-like cells and/or progenitor cells may be used for identification of patients suffering from glioblastoma multiforme (GBM).
  • GBM glioblastoma multiforme
  • Patients suffering from GBM have a very bad prognosis and early identification of such patients would be highly beneficial.
  • the patients identified to be suffering from GBM may be treated as described herein below in the section "Method of treatment”. Identification of VEGFR2 antagonist
  • VEGFR2 can be used for identifying a compound capable of inhibiting cancer stem-like cells and/or progenitor cells expressing VEGFR2 through binding to VEGFR2, said method comprising contacting VEGFR2 with a test compound and detecting autophosphorylation of VEGFR2 and/or activation of c- Raf/MAPK, P13K/Akt and/or PLCyl/PKC pathway and selecting the compounds which inhibits the activation of said pathways.
  • VEGFR2 are expressed on cells or cell lines and said cells or cell lines are then contacted with the test compound and autophosphorylation of VEGFR2 and/or activation of c- Raf/MAPK, P13K/Akt and/or PLCyl/PKC pathway is detected and compounds which inhibits the activation of said pathways are selected.
  • the cell or cell line may be a primary cell, a primary cell culture or an established cell line.
  • the cell line may for example be selected form the group consisting of U1 18MG, U87MG, T98G, A172, Gli-06, SF126, U373MG and U251 MG.
  • the cells may for example be selected from the group consisting of primary cells from Glioblastoma Multiforme, primary cells from patients diagnosed with cancer, primary cell lines and primary xenograft cells.
  • a reporter gene of the present invention is a gene construct with a promoter operatively linked to VEGFR2 said promoter initiating the expression of an enzyme or other compound (reporter gene product) which induce visually identifiable characteristics.
  • a reporter gene product could be fluorescent and luminescent proteins, such as for example Green Fluorescent Protein (GFP) and luciferase, respectively.
  • the activation of VEGFR2 is then measured by the activation of the reporter gene and hence the formation of gene product visualized in one embodiment as fluorescent protein, which can be observed using standard methods known in the art to observe fluorescence.
  • the detection of activation is done by Western blot analysis.
  • the VEGFR2 antagonist identified is able to pass the cellular membrane of cancer stem-like cells and/or progenitor cells as well as the blood-brain barrier. Antagonists
  • the present invention relates to the field of diagnosis and treatment of hyperproliferative diseases, such as cancer.
  • the invention relates to methods of identifying and killing cancer stem-like cells and/or progenitor cells expressing VEGFR2 using compounds capable of associating with VEGFR2 and in particular VEGFR2 antagonists and/or NRP-1 antagonists.
  • the invention relates to identification of compounds useful for identifying and killing cancer stem-like cells and/or progenitor cells, said compounds being VEGFR2 antagonists.
  • An antagonist of the present invention could also be directed at the native ligand for VEGFR2 (e.g. VEGF), however it is preferred that the antagonist of the present invention is directed at the receptor VEGFR2 as receptor specific antagonists are more effective and independent of receptor activation.
  • the VEGFR2 also generates VEGF upon activation and targeting VEGF would hence require a higher amount of antagonist to achieve the same IC 50 value (described below in this section "antagonists").
  • the antagonist is targeting VEGFR2.
  • NRP-1 neuropilin-1
  • NRP-1 is a isoform-specific receptor of VEGF, which function as co-receptor with VEGFR2 (see figure 12). Inhibiting NRP-1 would indirectly inhibit the activity of VEGFR2.
  • a NRP-1 antagonist may be a small molecule, peptides and/or proteins.
  • an antagonist targeting NRP-1 may be administered alone or in combination with antagonist targeting VEGFR2 directly.
  • the NRP-1 antagonist and the VEGFR2 antagonist is administered in combination.
  • a VEGFR2 antagonist according to the present invention is a type of VEGFR2 ligand or drug that does not provoke a biological response itself upon binding to VEGFR2, but blocks or dampens agonist-mediated responses.
  • antagonists have affinity but lower efficacy for VEGFR2, preferably the antagonist of the present invention have low efficacy for VEGFR2, more preferably no efficacy for VEGFR2.
  • a molecule with efficacy to the VEGFR2 is a molecule which can activate the downstream pathways linked to said receptor. Said pathways are the c- Raf/MAPK, P13K/Akt and/or PLCyl/PKC pathway.
  • VEGFR2 antagonist Binding of the VEGFR2 antagonist will disrupt the interaction and inhibit the function of an agonist (such as VEGF) or inverse agonist at receptors.
  • Antagonists may mediate their effects by binding to the active site or to allosteric sites on receptors, or they may interact at unique binding sites not normally involved in the biological regulation of the receptor's activity. Antagonist activity may be reversible or irreversible depending on the longevity of the antagonist-receptor complex, which, in turn, depends on the nature of antagonist receptor binding. The majority of drug antagonists achieve their potency by competing with endogenous ligands or substrates at structurally-defined binding sites on receptors.
  • the antagonists of the present invention should be able to pass the cellular membranes of cancer stem-like cells and/or progenitor cells as well as the blood-brain barrier of a human subject.
  • the potency of an antagonist is usually defined by its IC 50 value. This can be calculated for a given antagonist by determining the concentration of antagonist needed to elicit half inhibition of the maximum biological response of an agonist. Elucidating an IC 50 value is useful for comparing the potency of drugs with similar efficacies; however the dose-response curves produced by both drug antagonists must be similar. The lower the IC-50, the greater the potency of the antagonist and the lower the concentration of drug that is required to inhibit the maximum biological response. Lower concentrations of drugs may be associated with fewer side-effects.
  • the biological response is evaluated by determination of activation of downstream pathways linked to the VEGFR2, such as the c-Raf/MAPK, P13K/Akt and/or PLCyl/PKC pathway.
  • the activation of said pathways can be measured by methods known to the person skilled in the art.
  • the IC 50 value of the antagonists for the VEGFR2 is below 50 ⁇ , more preferably below 40 ⁇ , such as below 30 ⁇ , more preferably below 20 ⁇ , such as below 10 ⁇ , more preferably below 5 ⁇ , such as below 2 ⁇ , more preferably 1 ⁇ , such as 0.5 ⁇ , preferably 100nM, preferably below 90nM, such as below 80 nM, more preferably below 70 nM, such as below 60 nM, more preferably below 50 nM, such as below 40 nM, more preferably below 30 nM, such as below 20 nM, more preferably below 10 nM, such as below 5 nM, more preferably below 4 nM, such as below 3 nM, more preferably below 2 nM, such as below 1 nM, more preferably below 0.9 nM, such as below 0.8 nM, more preferably below 0.7 nM, such as below 0.6 nM, more preferably below
  • the IC 50 value is below 1 nM, more preferably below 0.9 nM, such as below 0.8 nM, more preferably below 0.7 nM, such as below 0.6 nM, more preferably below 0.5 nM, such as below 0.4 nM, more preferably below 0.3 nM, such as below 0.2 nm, more preferably below 0.1 nM.
  • the antagonist can be a competitive antagonist (also known as surmountable antagonist), which reversibly bind to receptors at the same binding site (active site) as the endogenous ligand or agonist, but without activating the receptor. Agonists and antagonists "compete" for the same binding site on the receptor. Once bound, an antagonist will block agonist binding.
  • the level of activity of the receptor will be determined by the relative affinity of each molecule for the site and their relative concentrations. High concentrations of a competitive agonist will increase the proportion of receptors that the agonist occupies, and higher concentrations of the antagonist will be required to obtain the same degree of binding site occupancy.
  • the antagonist can be a non-competitive antagonist (sometimes called non-surmountable antagonist), which is an allosteric antagonist.
  • non-competitive antagonists bind to a distinctly separate binding site from the agonist, exerting their action to that receptor via the other binding site.
  • Non-competitive antagonists do not compete with agonists for binding.
  • the bound antagonists may prevent conformational changes in the receptor required for receptor activation after the agonist binds. No amount of agonist can completely overcome the inhibition once it has been established.
  • depression of the maximal response of agonist dose-response curves, and in some cases, rightward shifts is produced. The rightward shift will occur as a result of a receptor reserve and inhibition of the agonist response will only occur when this reserve is depleted.
  • Uncompetitive antagonists differ from non-competitive antagonists in that they require receptor activation by an agonist before they can bind to a separate allosteric binding site. This type of antagonism produces a kinetic profile in which the same amount of antagonist blocks higher concentrations of agonist better than lower concentrations of agonist.
  • the antagonist of the present invention can be competitive, non-competitive and/or uncompetitive.
  • the antagonists of the present invention can be biologically active peptide that binds to VEGFR2, an anti-VEGFR2 antibody and/or a biologically active molecule that binds to the tyrosine kinase domain of VEGFR2.
  • the antagonist of the present invention binds to the tyrosine kinase domain of VEGFR2 and/or to the kinase domain of a functional variant of VEGFR2 (as described in the section "VEGFR2").
  • VEGFR2 antagonist is of the formula (I):
  • X 2 is C or N
  • X 3 is C or N
  • R 2 is -O- CH 3
  • R 3 is -O- R 8
  • R 4 is -C- heteroaromatic ring structure
  • R 5 is aromatic or heteroaromatic ring structure or ring system, optionally substituted with one or more substituent's selected from the group consisting of halogen, short alkyl, amide and - NH - C(O) - NH - R 7
  • R 6 is aromatic ring structure optionally substituted with halogen
  • R 7 is heteroaromatic ring optionally substituted with short alkyl.
  • R 8 is short alkyl optionally substituted with heterocycle, said heterocycle optionally substituted with short alkyl
  • X 3 is N, and when X 2 is N, then X 3 is also N.
  • R 5 is a benzyl ring.
  • Short alkyl as used herein unless otherwise indicated is an alkyl chain of 1 -6 carbon atoms, preferably 1 -3 carbon atoms more preferably one carbon. In one embodiment of the invention the preferred short alkyl is methyl. In another embodiment of the present invention the VEGFR2 antagonist is of the formula (II):
  • X is N or C optionally substituted with R 3
  • Ri is selected from the group consisting ofshort alkyl and alkenyl, optionally substituted with aromatic or heteroaromatic ring structure, said heteroaromatic ring structure optionally substituted with -C(O) - NH - C - C - NH(CH 3 ) 2
  • R 2 is amide optionally substituted with aromatic or heteroaromatic ring structure optionally substituted with -NH-R 4
  • R 3 is selected from the group consisting of -OH and short alkyl.
  • R 4 is aromatic ring optionally substituted with short alkyl or -S(0) 2 - NH 2
  • said heteroaromatic ring structure of R 2 is a benzyl ring.
  • the VEGFR2 antagonist is of the formula (I I I):
  • Ri is an amide optionally substituted with aromatic or heteroaromatic ring structure, optionally substituted with halogen or triflourmethyl.
  • R 2 is -O -R 3 .
  • R 3 is an aromatic or heteroaromatic ring structure optionally substituted with - C(O) -
  • VEGFR2 antagonist is selected from the group consisting of:
  • VEGFR2 antagonist is selected from the group consisting of:
  • VEGFR2 antagonsit is selected from the group consisting of:
  • VEGFR2 antagonist is selected from the group consisting of:
  • the antagonist of the present invention is selected from the group consisting of ZM323881 , DC101 , MAB3572, AF357, GW654652, GW612286, GW695612, SU5416, SU1498, SU6668, SU5402, Src 11 , Pazopanib, Axtinib, Sorafenib, PTK787/ZK222584, AV951 , Cediranib, Sunitinib, and vandetanib, and other antagonists with same function and structure as these, including pro-drugs, crystals, solvates, salts and esters thereof.
  • the antagonist is a functional homologue of these antagonists.
  • the antagonist of the present invention is selected from the group consisting of ZM323881 , DC101 , MAB3572, AF357, GW654652, GW612286, GW695612, SU5416, SU1498, SU6668, SU5402, Src 11 , Pazopanib, Axtinib, Brivanib alaninate, Pazopanib, Sorafenib, PTK787/ZK222584, AV951 , Cediranib, Sunitinib, Tivozanib, Vatalanib and vandetanib, and other antagonists with same function and structure as these, including pro-drugs, crystals, solvates, salts and esters thereof.
  • the antagonist is a functional homologue of these antagonists.
  • VEGFR inhibitors described in the following patents and patent applications can be used in the present invention: US 6,563,618, US 2003/016601 1 , US 2006/0223133, PCT/JP 1998/05697, US 2006/0241 1 15, WO 2005/070891 , US 6,258,812, US 2003/0105091 , WO 01 /37820, US 6,235,764, WO 01 /32651 , US 6,630,500, US 6,515,004, US 6,713,485, US 5,521 ,184, US 5,770,599, US 5,747,498, WO 02/68406, WO 02/66470, WO 02/55501 , WO 04/05279, WO 04/07481 , WO 04/07458, WO 06/012374, WO 06/1 16713, WO 04/09784, WO 02/591 10, WO 99/45009, WO 00/59509, WO 99/61422, US 5,990,141 , WO
  • the VEGFR2 antagonist is an anti-VEGFR2 antibody.
  • the molecule binding the cancer stem-like cells according to the present invention is an antibody.
  • Said antibody can be any suitable antibody known in the art.
  • the antagonist of the present invention is a binding partner such as immunologically active fragments of antibodies or single chain antibodies.
  • Antibody molecules are typically Y-shaped molecules whose basic unit consists of four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked by disulfide bonds. Each of these chains is folded in discrete domains.
  • the C-terminal regions of both the heavy and light chains are conserved in sequence and are called the constant regions, also known as C-domains.
  • the N-terminal regions, also known as V-domains are variable in sequence and are responsible for the antibody specificity.
  • the antibody specifically recognizes and binds to an antigen mainly through six short complementary-determining regions located in their V- domains.
  • the VEGFR2 antagonist is a binding partner, such as a fragment of an anti-VEGFR2 antibody, preferably an antigen binding fragment or variable region.
  • antibody fragments useful with the present invention include Fab, Fab', F(ab') 2 and Fv fragments.
  • Papain digestion of antibodies produces two identical antigen bindig fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily.
  • Pepsin treatment yields an F(ab') 2 fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc').
  • Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
  • the antibody fragments Fab, Fv and scFv differ from whole antibodies in that the antibody fragments carry only a single antigen-binding site.
  • Recombinant fragments with two binding sites have been made in several ways, for example, by chemical cross-linking of cysteine residues introduced at the C-terminus of the VH of a Fv (Cumber et al., 1992), or at the C-terminus of the VL of an scFv (Pack and Pluckthun, 1992), or through the hinge cysteine residues of Fab's (Carter et al., 1992).
  • Preferred antibody fragments retain some or essential all the ability of an antibody to selectively binding with its antigen or receptor.
  • Preferred fragments of the present invention are defined as follows:
  • Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule.
  • a Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain.
  • Fab' is the fragment of an antibody molecule and can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule.
  • Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
  • (Fab')2 is the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction.
  • F(ab')2 is a dimer of two
  • Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non- covalent association (VH -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH -V L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
  • the binding partner is a single chain antibody ("SCA"), defined as a genetically engineered molecule comprising the variable region of the light chain, and the variable region of the heavy chain, linked by a suitable poly-5 peptide linker as a genetically fused single chain molecule.
  • SCA single chain antibody
  • the SCA consists of only part of the variable region of the light chain, and part of the variable region of the heavy chain, linked by a suitable poly-5 peptide linker as a genetically fused single chain molecule.
  • the SCA consists of the variable region of the light chain, and the variable region of the heavy chain, linked by a suitable poly-5 peptide linker as a genetically fused single chain molecule.
  • single chain antibodies are also referred to as “single-chain Fv” or “sFv” antibody fragments.
  • the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
  • the antibody fragments to be used with the invention may be produced in any suitable manner known to the person skilled in the art.
  • Several microbial expression systems have already been developed for producing active antibody fragments, e.g. the production of Fab in various hosts, such as E. coli, yeast, and the filamentous fungus Trichoderma reesei are known in the art.
  • the recombinant protein yields in these alternative systems can be relatively high. 1 -2 g/l for Fab secreted to the periplasmic space of E. coli in high cell density fermentation or at a lower level, e.g.
  • the fragments can be produced as Fab's or as Fv's, but additionally it has been shown that a VH and a VL can be genetically linked in either order by a flexible polypeptide linker, which combination is known as an scFv.
  • the antibody is preferably selected from the group consisting of a monoclonal antibody, a fragment of an antibody, a derivative of an antibody, a chimerized antibody, a humanized antibody and a single chain antibody.
  • Said antibody of the present invention should function as a VEGFR2 antagonist and demonstrate the same preferred IC50 values as described above in this section.
  • the antagonist of the present invention is a NRP-1 antagonist.
  • the NRP-1 antagonist may be a small molecule, a peptide and/or a protein and may be selected from the group consisting of EG01257, Anti-neuropilin-1 , Anti-NRP1 , M 1685A, M1685A, MNRP 1685A, MNRP1685A, R 7347, R7347 and RG7347, and other antagonists with same function and structure as these, including pro-drugs, crystals, solvates, salts and esters thereof.
  • the antagonist is a functional homologue of these antagonists.
  • NRP-1 antagonist is of the formula (IV):
  • W is arylene, heteroarylene or Formula (V)
  • each X is independently an N-containing heteroarylene, N-containing cycloalkylene or NR;
  • Y is N-containing heteroaryl, N-containing cycloalkyl, NR2, OR1 , CN or C02R;
  • R is H or C1 -C6 alkyl
  • R1 is H, C1 -C6 alkyl or an amino acid
  • n 2, 3, 4 or 5;
  • m 1 , 2 or 3.
  • NRP-1 antagonist is of the formula
  • each X is independently an N-containing heteroarylene, N-containing cycloalkylene or NR;
  • Y is N-containing heteroaryl, N-containing cycloalkyl, NR, OR1 , CN or C02R;
  • R is H or C1 -C6 alkyl
  • R1 is H, C1 -C6 alkyl or an amino acid
  • n O, 1 , 2, 3, 4 or 5;
  • m 1 , 2 or 3.
  • the NRP-1 antagonist is selected from the group consisting of EG01257, Anti-neuropilin-1 , Anti-NRP1 , M 1685A, M1685A, MNRP 1685A, MNRP1685A, R 7347, R7347 and RG7347.
  • the molecule that specifically binds VEGFR2 (preferably a VEGFR2 antagonist, such as a VEGFR2 antibody) or a synthetic probe is directly labelled with a detectable label, wherein the detectable label preferably may be selected from the group consisting of fluorochromes, colour agents, heavy metals, enzymes and radioactive labels. Said labels are preferably covalently attached thereto.
  • the VEGFR2 antagonist of the present invention is labelled with fluorochromes.
  • the antagonist described in the section "antagonist” are labelled. Said antagonist may be used in the present invention as described in the section "method of detecting cancer stem-like cells and/or progenitor cells".
  • probes or antagonists to VEGFR2 on or in the cell may be observed under a fluorescence microscope as a bright fluorescence or may be detected by a fluorimetric apparatus.
  • the probes or antagonists are indirectly labelled with biotin or other binding partners or enzymes for example, such as alkaline phosphatase or peroxidase.
  • a binding partner is a molecule which is binding the VEGFR2 antagonist with high affinity or is linked to the VEGFR2 antagonist, and therefore can be used for detection of the binding of the VEGFR2 antagonist to VEGFR2.
  • the antagonist is indirectly labelled with biotin.
  • Biotin may be detected using suitable streptavid in/avid in molecules known to people skilled in the art. These complexes may comprise fluorochromes or suitable enzymes.
  • two or more selective labellings may be performed.
  • the two or more labellings may be a combination of any of the labellings used for single labelling described above. Accordingly, the combined labelling may be carried out by the use of two or more different hybridisation probes.
  • a combination of different antagonists may be used, either with specificity for the same antigen or with specificity for different antigens.
  • labelling with a combination of nucleotide probes and antagonists may be performed.
  • a combination of a labelled VEGFR2 antagonist as described in the section "antagonist” and a labelled antibody for another antigen other than VEGFR2 may be used as described in the section "method of detecting a cancer stem-like cell and/or progenitor cells”.
  • the selectively labelled cells may be isolated from the cell sample and processed further.
  • the isolated cancer stem-like cells and/or progenitor cells expressing VEGFR2 may be used for diagnosis or as marker of the presence of cancer stem-like cells and/or progenitor cells expressing VEGFR2 as described in the section "method of detecting a cancer stem-like cell and/or progenitor cells".
  • the invention also relates to embodiments wherein other molecules than VEGFR2 antagonists are labelled with a detectable label, e.g. molecules specifically binding CD31 , Tusc5, 7B4, BNH9/BNF13, CD105, CD146, D2-40, EN4, PAL-E, CD133 or integrin Alpha-6. These molecules may also be labelled with any of the detectable labels described in this section.
  • a detectable label e.g. molecules specifically binding CD31 , Tusc5, 7B4, BNH9/BNF13, CD105, CD146, D2-40, EN4, PAL-E, CD133 or integrin Alpha-6.
  • the present invention relates in one embodiment to the combination of administration of a VEGFR2 antagonist and/or a NRP-1 antagonist as described in the section "antagonist" and radiation therapy.
  • Radiation therapy also denoted radiotherapy and/or radiation oncology, and sometimes abbreviated to XRT, according to the present invention is the medical use of ionizing radiation (IR) as part of cancer treatment to control malignant cells (not to be confused with radiology, the use of radiation in medical imaging and diagnosis).
  • Radiation therapy may be used for curative or adjuvant treatment. It may be used as palliative treatment (where cure is not possible and the aim is for local disease control or symptomatic relief) or as therapeutic treatment (where the therapy has survival benefit and it can be curative).
  • Radiotherapy may be used for the treatment of tumours (benign as well as malignant), and may be used as a primary or adjuvant modality. It is also common to combine radiotherapy with surgery, cytotoxic drugs, hormone therapy or some mixture of the three. Most common tumour types can be treated with radiotherapy in some way.
  • the precise treatment intent (curative, adjuvant, neoadjuvant, therapeutic, or palliative) will depend on the tumour type, location, and stage, as well as the general health of the patient.
  • shaped radiation beams may in the present invention be aimed from several angles of exposure to intersect at the tumour, providing a much larger absorbed dose there than in the surrounding, healthy tissue.
  • Radiation therapy works by damaging the DNA of cells.
  • the damage is caused by a photon, electron, proton, neutron, or ion beam directly or indirectly ionizing the atoms which make up the DNA chain. Indirect ionization happens as a result of the ionization of water, forming free radicals, notably hydroxyl radicals, which then damage the DNA.
  • free radicals notably hydroxyl radicals
  • most of the radiation effect is through free radicals.
  • cells have mechanisms for repairing DNA damage, breaking the DNA on both strands proves to be the most significant technique in modifying cell characteristics.
  • cancer cells generally are undifferentiated and stem cell-like, they reproduce more, and have a diminished ability to repair sub-lethal damage compared to most healthy differentiated cells.
  • the DNA damage is inherited through cell division, accumulating damage to the cancer cells, causing them to die or reproduce more slowly.
  • the amount of radiation used in radiation therapy is measured in gray (Gy), and varies depending on the type and stage of cancer being treated.
  • radiation therapy is given for curative cases, and the typical dose for a solid epithelial tumour ranges from 60 to 80 Gy, while lymphomas are treated with 20 to 40 Gy.
  • radiation therapy is administered for preventative (adjuvant) purposes in which case the doses are typically around 45 - 60 Gy in 1 .8 - 2 Gy fractions (for Breast, Head and Neck cancers respectively.)
  • doses are typically around 45 - 60 Gy in 1 .8 - 2 Gy fractions (for Breast, Head and Neck cancers respectively.)
  • radiation oncologists determine whether the patient is receiving cytotoxic drugs, patient comorbidities, whether radiation therapy is being administered before or after surgery, and the degree of success of surgery.
  • the amount of radiation used is preferably in the ranges 80-100 Gy, more preferably 60-80 Gy, such as 60-70 Gy, more preferably 40-60 Gy, such as 40-50 Gy, more preferably 20-40 Gy, such as 20-30 Gy, more preferably 1 -20 Gy, such as 1 -10 Gy, more preferably 1 -2 Gy.
  • tumours in the head-and- neck demonstrate this behavior.
  • the total dose may be fractionated (spread out over time) for several important reasons. Fractionation allows normal cells time to recover, while tumour cells are generally less efficient in repair between fractions. Fractionation may also allow tumour cells that were in a relatively radio-resistant phase of the cell cycle during one treatment to cycle into a sensitive phase of the cycle before the next fraction is given. Similarly, tumour cells that were chronically or acutely hypoxic (and therefore more radioresistant) may reoxygenate between fractions, improving the tumour cell kill.
  • two fractions of radiation therapy per day are used near the end of a course of treatment. This schedule, known as a concomitant boost regimen or hyperfractionation, is used on tumours that regenerate more quickly when they are smaller. In particular, tumours in the head-and- neck demonstrate this behavior.
  • CHART Continuous Hyperfractionated Accelerated Radiotherapy
  • the fractionation schedule may preferably consists of 1 .8 to 2 Gy per day, for 3 to 7, preferably five days a week, more preferably 1 .5 to 1 .8 Gy per day, for 3 to 7, preferably five days a week.
  • brachytherapy uses sealed radioactive sources placed precisely in the area under treatment, and systemic radioisotopes are given by infusion or oral ingestion.
  • brachytherapy can use temporary or permanent placement of radioactive sources.
  • the temporary sources are usually placed by a technique called afterloading.
  • afterloading a hollow tube or applicator is placed surgically in the organ to be treated, and the sources are loaded into the applicator after the applicator is implanted.
  • Intraoperative radiotherapy or IORT is used, which is a special type of radiotherapy that is delivered immediately after surgical removal of the cancer. This method has been employed in breast cancer (TARGeted Introperative radioTherapy or TARGIT), brain tumours and rectal cancers.
  • any of the abovementioned radiation therapies are contemplated to be part of the invention.
  • radiation therapy may be used curative and palliative and in combination with the administration of a VEGFR2 antagonist as described in the section "antagonist".
  • a VEGFR2 antagonist as described in the section "antagonist" is administered immediately prior to the administration of radiation therapy, as described here above.
  • Immediately prior to radiation therapy as used herein denotes an administration 5 days of initiation of radiation therapy, more preferably with 4 days, such as within 3 days, more preferably within 2 days, such as with in 1 day, more preferably within 20 hours, such as within 10 hours, more preferably within 5 hours, more preferably within 4.5 hours, such as with in 4 hours, more preferably within 3.5 hours, such as within 3 hours, more preferably within 2.5 hours, such as within 2 hours, more preferably within 1 .5 hours, such as within 1 hour, more preferably within 0.5 hour, such as within 15 minutes, more preferably within 10 minutes, such as within 5 minutes of radiation therapy.
  • the VEGFR2 antagonist is administered within 2 hour of initiation of radiation therapy, such as within 1 hour, more preferably with 0.5 hour of initiation of radiation therapy.
  • a VEGFR2 antagonist as described in the section "antagonist" is administered prior to the administration of radiation therapy, as described here above.
  • Prior to radiation therapy as used herein denotes an administration within 3 weeks of initiation of radiation therapy, more preferably within 2 weeks, such as within 1 week, more preferably within 6 days, such as within 5 days, more preferably with 4 days, such as within 3 days, more preferably within 2 days, such as with in 1 day, more preferably within 20 hours, such as within 10 hours, more preferably within 5 hours, more preferably within 4.5 hours, such as with in 4 hours, more preferably within 3.5 hours, such as within 3 hours, more preferably within 2.5 hours, such as within 2 hours, more preferably within 1 .5 hours, such as within 1 hour, more preferably within 0.5 hour, such as within 15 minutes, more preferably within 10 minutes, such as within 5 minutes of radiation therapy.
  • the VEGFR2 antagonist is administered within 2 hour of initiation of radiation therapy, such as within 1 hour, more preferably with 0.5 hour of initiation of radiation therapy.
  • a NRP-1 antagonist as described in the section "antagonist" is administered immediately prior to the administration of radiation therapy, as described here above.
  • initiation of radiation therapy denotes an administration within 3 weeks of initiation of radiation therapy, more preferably within 2 weeks, such as within 1 week, more preferably within 6 days, such as within 5 days of initiation of radiation therapy, more preferably with 4 days, such as within 3 days, more preferably within 2 days, such as with in 1 day, more preferably within 20 hours, such as within 10 hours, more preferably within 5 hours, more preferably within 4.5 hours, such as with in 4 hours, more preferably within 3.5 hours, such as within 3 hours, more preferably within 2.5 hours, such as within 2 hours, more preferably within 1 .5 hours, such as within 1 hour, more preferably within 0.5 hour, such as within 15 minutes, more preferably within 10 minutes, such as within 5 minutes of radiation therapy.
  • the NRP-1 antagonist is administered within 2 hour of initiation of radiation therapy, such as within 1 hour, more preferably with 0.5 hour of initiation of radiation therapy.
  • a NRP-1 antagonist as described in the section "antagonist" is administered in combination with an anti-VEGF antibody (e.g. avastin) immediately prior to the administration of radiation therapy, as described here above.
  • an anti-VEGF antibody e.g. avastin
  • Immediately prior to radiation therapy as used herein denotes an administration within 3 weeks of initiation of radiation therapy, more preferably within 2 weeks, such as within 1 week, more preferably within 6 days, such as within 5 days of initiation of radiation therapy, more preferably with 4 days, such as within 3 days, more preferably within 2 days, such as with in 1 day, more preferably within 20 hours, such as within 10 hours, more preferably within 5 hours, more preferably within 4.5 hours, such as with in 4 hours, more preferably within 3.5 hours, such as within 3 hours, more preferably within 2.5 hours, such as within 2 hours, more preferably within 1 .5 hours, such as within 1 hour, more preferably within 0.5 hour, such as within 15 minutes, more preferably within 10 minutes,
  • the hyperproliferative disease to be treated or reducing the risk of recurrence of with the present invention is contemplated to be any disease originating in hyperproliferative cells present in the subject to be treated.
  • the hyperproliferative disease is a tumour of connective tissue origin, nervous system origin, and/or hematologic origin.
  • a tumour or tumor is the name for a neoplasm or a solid lesion formed by an abnormal growth of cells (termed neoplastic) which looks like a swelling. Tumour is not synonymous with cancer.
  • a tumour can be benign, pre-malignant or malignant, whereas cancer is by definition malignant. In the present apllication the words tumour and tumor are used interchangeable.
  • the hyperproliferative disease is a cancer of connective tissue origin, nervous system origin, and/or hematologic origin.
  • a cancer medical term: malignant neoplasm
  • malignant neoplasm is a class of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). These three malignant properties of cancers differentiate them from benign tumours, which are self-limited, and do not invade or metastasize. Most cancers form a tumour but some, like leukemia, do not.
  • the disease to be treated, prevented or reducing the risk of recurrence of is ovarian and/or prostate cancer.
  • the disease to be treated, prevented or reducing the risk of recurrence of is a tumour of connective tissue origin, nervous system origin, and/or hematologic origin.
  • the disease to be treated, prevented or reducing the risk of recurrence of is a cancer of connective tissue origin, nervous system origin, and/or hematologic origin.
  • the disease to be treated, prevented or reducing the risk of recurrence of is selected from the group consisting of head and neck tumours, Kaposi's sarcoma, CNS neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas, cerebral metastases, melanomas, gastrointestinal sarcomas, renal sarcomas, rhabdomyosarcomas, gliomas, glioblastomas, glioblastoma multiforme, leimyosarcomas, squamous cell carcinomas, basal cell carcinomas, human malignant keratinocytes, leukemias, multiple myelomas, lymphomas, acute myelocytic leukemia (AML), chronic myelocytic leukemia (CLL), erythrocytic leukemia, monocytic leukemia, Hodgkin's disease and Non-Hodgkin's disease.
  • the disease to be treated, prevented or reducing the risk of recurrence of is selected from the group consisting of glioma, glioblastoma, glioblastoma multiforme, astrocytoma, oligodendroglioma, ependymomas, medulloblastoma, meningioma, neurinoma and Schwannoma.
  • the disease to be treated, prevented or reducing the risk of recurrence of is glioblastomas or glioblastoma multiforme.
  • a pharmaceutical composition comprising, as an active ingredient, a VEGFR2 antagonist of the present invention together with a pharmaceutically acceptable carrier or diluent.
  • the antagonist of the invention may be administered alone or in combination with pharmaceutically acceptable carriers, diluents or excipients, in either single or multiple doses.
  • suitable pharmaceutical acceptable carriers, diluents and excipients include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents.
  • compositions according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 21 st Edition, 2000, Lippincott Williams & Wilkins.
  • compositions formed by combining a VEGFR2 antagonist, or a pharmaceutically acceptable salt, solvate or prodrug thereof, with pharmaceutical acceptable carriers, diluents or excipients can be readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, suppositories, injectable solutions and the like.
  • the carrier is a finely divided solid such as talc or starch which is in a mixture with the finely divided active component.
  • the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • Suitable solid carriers include, but are not limited to, lactose, terra alba, sucrose, cyclodextrins (such as hydroxypropyl- -cyclodextrin, HPCD), talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose.
  • liquid carriers include, but are not limited to, syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, polysorbates (such as Tween-20 or Tween-80), Cremophor EL or water.
  • the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
  • sustained release material such as glyceryl monostearate or glyceryl distearate
  • nano- formulations such as nano-emulsion or nano-dispersions.
  • the excipients used in the pharmaceutical formulation conforms to the "Generally recognized as Safe" GRAS listing provided by the FDA.
  • compositions may be specifically formulated for administration by any suitable route such as the oral and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route or local injection. It will be appreciated that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient chosen.
  • compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders and granules. Where appropriate, they can be prepared with coatings such as enteric coatings or they can be formulated so as to provide controlled release of the active ingredient such as sustained or prolonged release according to methods well known in the art.
  • a VEGFR2 antagonist of the present invention may suitably be combined with an oral, non-toxic, pharmaceutically acceptable carrier such as ethanol, glycerol, water or the like.
  • an oral, non-toxic, pharmaceutically acceptable carrier such as ethanol, glycerol, water or the like.
  • suitable binders, lubricants, disintegrating agents, flavouring agents and colourants may be added to the mixture, as appropriate.
  • suitable binders include, e.g., lactose, glucose, starch, gelatin, acacia gum, tragacanth gum, sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes or the like.
  • Lubricants include, e.g., sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride or the like.
  • Disintegrating agents include, e.g., starch, methyl cellulose, agar, bentonite, xanthan gum, sodium starch glycolate, crospovidone, croscarmellose sodium or the like. Additional excipients for capsules include macrogols or lipids.
  • the active compound of formula (I) is mixed with one or more excipients, such as the ones described above, and other pharmaceutical diluents such as water to make a solid preformulation composition containing a homogenous mixture of a compound of the present invention.
  • the term "homogenous” is understood to mean that the compound of the present invention is dispersed evenly throughout the composition so that the composition may readily be subdivided into equally effective unit dosage forms such as tablets or capsules.
  • the preformulation composition may then be subdivided into unit dosage forms containing for example from about 0.05 to about 1000 mg, in particular from about 0.1 to about 500 mg, e.g.
  • a dosage unit of a formulation contain between 0.1 mg and 1000 mg, preferably between 1 mg and 100 mg, such as 5-50 mg of a compound of the invention.
  • Liquid formulations for either oral or parenteral administration of the VEGFR2 antagonist include, e.g., aqueous solutions, syrups, elixirs, aqueous or oil suspensions and emulsion with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil.
  • Suitable dispersing or suspending agents for aqueous suspensions include synthetic or natural gums such as tragacanth, alginate, acacia, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose or polyvinylpyrolidone.
  • compositions for parenteral administration include sterile aqueous and non-aqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use.
  • solutions containing a compound of this invention or a pharmaceutically acceptable salt, solvate or prodrug thereof in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solution may be employed.
  • Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration.
  • the oily solutions are suitable for intra-articular, intra-muscular and subcutaneous injection purposes.
  • the preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • parenteral administration for VEGFR2 antagonist of the invention, wherein the active part of the molecule contains acid labile groups, such as e.g. ester groups.
  • acid labile groups such as e.g. ester groups.
  • the formulations of a compound of the present invention may include one or more additional ingredients such as diluents, buffers, flavouring agents, colourant, surface active agents, thickeners, preservatives, e.g. methyl hydroxybenzoate (including anti-oxidants), emulsifying agents and the like.
  • additional ingredients such as diluents, buffers, flavouring agents, colourant, surface active agents, thickeners, preservatives, e.g. methyl hydroxybenzoate (including anti-oxidants), emulsifying agents and the like.
  • a suitable dosage of the compound of the invention will depend on the age and condition of the patient, the severity of the disease to be treated and other factors well known to the practicing physician.
  • the compound may be administered for example either orally, parenterally or topically according to different dosing schedules, e.g. daily or with intervals, such as weekly intervals.
  • a single dose will be in the range from 0.01 to 100 mg/kg body weight, preferably from about 0.05 to 75 mg/kg body weight, more preferably between 0.1 to 50 mg/kg body weight, and most preferably between 0.1 to 25 mg/kg body weight.
  • the compound may be administered as a bolus (i.e. the entire daily dosis is administered at once) or in divided doses two or more times a day. Variations based on the aforementioned dosage ranges may be made by a physician of ordinary skill taking into account known considerations such as weight, age, and condition of the person being treated, the severity of the affliction, and the particular route of administration.
  • the VEGFR2 antagonists of this invention are generally utilized as the free substance or as a pharmaceutically acceptable salt or ester thereof.
  • One example is an acid addition salt of a compound having the utility of a free base.
  • a VEGFR2 antagonist contains a free base such salts are prepared in a conventional manner by treating a solution or suspension of a free base of the antagonist with a chemical equivalent of a pharmaceutically acceptable acid, for example, inorganic and organic acids.
  • Physiologically acceptable salts of a compound with a hydroxy group include the anion of said compound in combination with a suitable cation such as sodium or ammonium ion.
  • VEGFR2 antagonist of the present invention with one or more other active compounds may be either concomitantly or sequentially.
  • the other active compound may be a NRP-1 antagonist.
  • the VEGFR2 antagonist of the invention may also be formulated in a pharmaceutical composition comprising one or more further active substances alone, or in combination with pharmaceutically acceptable carriers, diluents, or excipients in either single or multiple doses.
  • suitable pharmaceutical acceptable carriers, diluents and excipients are as described herein above, and the one or more further active substances may be any active substances, or preferably an active substance as described in the section "combination treatment" herein below.
  • the preferred administration is parenteral and/or oral.
  • the VEGFR2 antagonist is administered directly to the tumour site.
  • administration directly to the tumour site is done through a gliadel wafer.
  • Gliadel wafers for use in the present invention are small, dime-sized biodegradable polymer wafers that are designed to deliver VEGFR2 antagonist directly into the surgical cavity created when a brain tumour is resected. Gliadel wafers are commercially available and can be purchased from Eisai Inc. (www.gliadel.com).
  • a compound of the present invention may also be used to advantage in combination with one or more other anti-proliferative or anti-neoplastic agents.
  • antiproliferative agents include, but are not limited to aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule active agents; alkylating agents; histone deacetylase inhibitors; compounds which induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antineoplastic antimetabolites; platin compounds; compounds targeting/decreasing a protein or lipid kinase activity and further anti-angiogenic compounds; compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase; gonadorelin agonists; anti-androgens; angiostatic steroids; methionine aminopeptidase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparanase inhibitor
  • a compound of the present invention may also be used to advantage in combination with known therapeutic processes, e.g., the administration of hormones or tumour cell damaging approaches, especially ionizing radiation.
  • a compound of the present invention may also be used as a radiosensitizer, including, for example, the treatment of tumours which exhibit poor sensitivity to radiotherapy.
  • a compound of the present invention may also be given in combination with one or more radiosensitizing drugs.
  • a radiosensitizing drug used in the present invention is a drug which makes tumours more sensitive to radiation therapy.
  • radiosensitizing drugs include but are not limited to; Cisplatin, Nimorazole and Cetuximab.
  • the VEGFR2 antagonist is administered in combination with Cisplatin and/or Nimorazole and/or Cetuximab.
  • the VEGFR2 antagonist and the radiosensitizing drug may be administered simultaneously or sequential as a combined medicament or as discrete entities.
  • the radiosensitizing drug and the VEGFR2 antagonist is formulated as a combined medicament.
  • combination is meant either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where a compound of the present invention and a combination partner may be administered independently at the same time or separately within time intervals that especially allow that the combination partners show a cooperative, e.g., synergistic, effect, or any combination thereof.
  • Method of treatment is meant either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where a compound of the present invention and a combination partner may be administered independently at the same time or separately within time intervals that especially allow that the combination partners show a cooperative, e.g., synergistic, effect, or any combination thereof.
  • the present invention relates to a method of treating or preventing diseases in a subject, said method comprises administering to said subject a therapeutically effective amount of a VEGFR2 antagonist, or pharmaceutically acceptable salts, solvates or prodrugs thereof, as defined herein, to a subject in need of such treatment.
  • the disease may be any disease or disorder as mentioned herein, such as for example mentioned in the section "hyperproliferative diseases", and the VEGFR2 antagonist may be administered alone or in a pharmaceutical composition, such as for example mentioned in the section "Pharmaceutical compositions".
  • preventing the disease is linked to reducing the risk of recurrence as described above. By reducing the risk of recurrence of the disease, the disease is in one embodiment prevented.
  • the administration of VEGFR2 antagonist according to the present invention can prevent the occurrence of the disease.
  • the method is a method of treating or preventing a hyperproliferative disease in a subject, said method comprises administering to said subject a therapeutically effective amount of a VEGFR2 antagonist of the present invention, or pharmaceutically acceptable salts, solvates or prodrugs thereof, as defined herein, to a subject in need of such treatment.
  • the hyperproliferative disease may be any hyperproloferative disease as described herein above.
  • the hyperproliferative disease is glioblastoma or glioblastoma multiforme.
  • the VEGFR2 antagonist of the present invention is administered in combination with one or more further active substances.
  • the active substances may be any active substances, and preferably an active substance as described herein above in the section "combination treatment". More preferably the one or more additional active substances are selected from the group consisting of antiproliferative, anti-neoplastic agents or radiosensitizing drugs.
  • the VEGFR2 antagonist of the present invention is administered in combination with ionizing radiation therapy.
  • the radiation therapy may be any form of radiation, and preferably ionizing radiation as described herein above in the section "radiation therapy”.
  • the compound of the present invention is administered to a subject immediately prior to radiation therapy.
  • the method of treatment or prevention of a hyperproliferative disease according to the invention comprises administration of the VEGFR2 antagonist of the present invention, or pharmaceutically acceptable salts, solvates or prodrugs thereof, as defined herein, in combination with administration of VEGF antibodies.
  • the compound of the present invention is administered to a subject prior to administration of anti-VEGF antibodies.
  • the method of treatment or prevention of a hyperproliferative disease according to the invention may consist of multiple treatments administered to the same subject.
  • Glioma cell populations enriched or depleted in cancer stem-like cells were isolated from human glioma surgical specimens. Briefly, tumours were immediately dissected with removal of gross necrosis; washed in Earle's balanced salt solution; subjected to a papain digestion followed by trituration, filtering, and lysis of RBC in PBS/water (1 :3) solution; and then cultured overnight (12 h) in neurobasal medium (with B27 and epidermal growth factor and basic fibroblast growth factor at 20 ng/mL) for recovery of cellular surface antigens before cell sorting. Primary glioma tumour samples were obtained from patients undergoing resection.
  • Fluorescence-activated cell sorting analysis Because papain digestion may cause loss of some cellular surface antigens such as CD133, it is critical to allow the isolated total tumour cells to reexpress their surface antigens in the neurobasal medium overnight (12 h) before sorting for CD133 + glioma cells.
  • Tumour cell cultures were subjected to fluorescence-activated cell sorting (FACS) analysis and cell sorting after the antigen recovery period.
  • FACS fluorescence-activated cell sorting
  • Human-specific anti-VEGFR2 (Biolegend) conjugated to AlexaFluor 647 conjugate was used for cell sorting and FACS analysis.
  • In vivo tumour formation assays VEGFR2 positive (top 10%) and negative (bottom 10%) glioma cells were transplanted into athymic BalbC nu/nu mice through intracranial or s.c. injection (figure 7).
  • Immunofluorescent staining Frozen human sections from glioblastoma multiforme were fixed with 4% paraformaldehyde, washed with TBS, incubated with anti-VEGFR2 monoclonal antibody (AbCam) and rabbit CD133 (AbCam) or CD31 (AbCam), and FITC-conjugated donkey anti-mouse IgG or anti-rabbit secondary antibody. Cells were co-stained with 4',6-diamidino-2-phenylindole (DAPI) and mounted with the anti-fade medium. Stained cells were examined under a fluorescent microscope (Zeiss Axiovert 200). Cell growth curves and inhibitor testing.
  • Cells were aliquoted into a 96-well plate at 1 ,000 or 4000 cells per well in duplicate. Cell growth was measured at the day 1 , 3, 5 and 7, and the effect of inhibitor was measured at 24, 48, 72, 96 hours after SU1498 or SU5416 treatment using the CellTiter-Glo assay kit (Promega, Madison, Wl, http://www.promega.com).
  • Ionizing radiation treatment in enrichment experiments of freshly dissociated human specimens and xenografts Freshly isolated ells were treated with ionizing radiation of 8 Gy (dose rate 2.18 Gy/min by an X-ray generator, Pantak, Berkshire, United Kingdom; HF160; 150 kV; 15mA) after 1 hour pre-treatment with the inhibitor SU1498 or left untreated, and analyzed 24, 48 and 72 hours after irradiation for the presence of CD133- and VEGFR2-positive cells using FACS Calibur (BD Biosciences).
  • mice In vivo experiments in mice.
  • Experiment 1 Injection of freshly dissociated cells from human biopsy or mouse xenograft that will have VEGFR2-knockdown using lentiviral shRNA. Two groups of mice will be injected:
  • mice Intracranial injection of 100 000 VEGFR2 negative cells and 5, or 8 days later, mice will be sacrificed and brain will be analyzed for presence of human VEGFR2 expressing cell - this is in order to evaluate re-expression of VEGFR2 due to stimuli from microenvironment.
  • mice will be divided into 4 groups:
  • mice will be treated with equal doses of IR and tumour formation will be followed. We expect mice with VEGFRshRNA to be significantly delayed in tumour formation.
  • GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEV

Abstract

The present invention relates to the field of diagnosis and treatment of hyperproliferative diseases, such as cancer. In particular, the invention relates to methods of identifying and killing cancer stem-like cells and/or progenitor cells using compounds capable of associating with VEGFR2 and/or NRP-1. Furthermore, the invention relates to identification of compounds useful for identifying and killing cancer stem-like cells and/or progenitor cells, said compounds being VEGFR2 antagonists and/or NRP-1 antagonists. The present invention does also relate to identification of patients that are sensitive to VEGFR2 antagonist and hence would benefit from treatment according to the present invention

Description

Targeting of VEGFR2
All patent and non-patent references cited in the application are hereby incorporated by reference in their entirety.
Field of invention
The present invention relates to the field of diagnosis and treatment of
hyperproliferative diseases, such as cancer. In particular, the invention relates to methods of identifying and killing cancer stem-like cells using compounds capable of associating with VEGFR2. Furthermore, the invention relates to identification of compounds useful for identifying and killing cancer stem-like cells and/or progenitor cells, said compounds being VEGFR2 antagonists. The present invention does also relate to identification of patients that are sensitive to VEGFR2 antagonist and hence would benefit from treatment according to the present invention.
Background of invention
Brain tumours strike deep into the psyche of those receiving and those delivering the diagnosis. Malignant astrocytomas, the most common subtype of primary brain tumours, are aggressive, highly invasive, and neurologically destructive tumours considered being among the deadliest of human cancers (Maher EA, Furnari, FB, Bachoo RM, Rowitch DH, Louis DN, Cavenee WK, DePinho R. Malignant glioma: genetics and biology of grave matter. Genes & Development. 2001 ; 15: 131 1 -1332.). Standard chemotherapy approaches have only resulted in modest improvements in time to progression and survival for patients with brain tumours (reviewed in Newton HB. Molecular neuro-oncology and development of targeted therapeutic strategies for brain tumours. Part 2: Pi3K/Akt/PTEN, mTOR, SHH/PCTH and angiogenesis. Expert Rev Anticancer Ther. 2004; 4: 105-128 and Reardon DA, Rich JN, Friedman HS, Bigner DD. Recent Advances in the Treatment of Malignant Astrocytoma. J Clin Oncol. 2006; 24: 1253-1265). The commonly used drugs are non-specific and unable to significantly modify the transformed phenotype of malignant brain tumour cells. Due to limitations to traditional chemotherapy, researchers have begun to investigate the development of more specific, targeted treatment modalities that exploit the molecular pathogenesis of cancer. Malignant astrocytomas (grade 11 l/l V), like other solid tumours, have extensive areas of hypoxia and necrosis. One of the key features of astrocytomas, affecting their biological and clinical behaviour, is dense vascularisation. The characteristic vascular morphology in high grade astrocytomas has led to the hypothesis that the formation of new blood vessels or angiogenesis is crucial to their growth. VEGF is the key factor involved in angiogenesis. Beside its pro-angiogenic function, VEGF also increases vascular permeability and was found to be over-expressed in a variety of tumours including highly vascularized and infiltrative astrocytomas (grade lll/IV), where VEGF expression correlates with tumour progression and poor prognosis. Interactions between tumour cells and their microenvironment are critically important in the biology of cancer, and include growth factor signalling in paracrine and autocrine manners. The bulk of current knowledge about the biology of VEGF and its receptors derives from studies of endothelial cells and VEGF's paracrine effects when secreted by diverse tumour cell types. Given the crucial role of angiogenesis and tumour spread in astrocytomas, the present invention demonstrates new findings in the relation between VEGF and tumour survival and recurrence after treatment.
VEGF and its receptors
Angiogenesis, a process highly regulated by a large number of pro- and anti- angiogenic factors, is essential not only in early embryogenesis but also in tumour development and progression. Angiogenesis also takes place during tissue growth and repair; female reproductive cycle, foetal development and inflammation. A wide range of pro- and anti-angiogenic factors are deregulated in brain tumours and released after a variety of physiological and pathological stimuli. Localized breakdown of extracellular matrix (ECM) precedes the proliferation, migration and tissue infiltration of endothelial cells. In time these cells remodel back into capillary structures, and a new ECM is deposited.
One of the best characterized pro-angiogenic factors is VEGF. VEGF is a member of VEGF protein family encompassing related growth factors, including VEGF B, C, D, PIGF (placenta growth factor) and the orf virus-encoded factor VEGF E. Six different isoforms of VEGF (ranging from 121 to 206 amino acid residues in size), the result of alternative splicing, have been identified in human (Robinson CJ, Stringer SE. The splice variants of vascular endothelial growth factor (VEGF) and their receptors. J Cell Sci. 2001 ; 1 14: 853-65). VEGF has been highlighted as a multi-functional chemokine stimulating differentiation, survival, proliferation, migration, tubulogenesis and vascular permeability in endothelial cells. It is required for normal development of blood vessels, and targeted inactivation of only a single allele of the VEGF during embryogenesis in mice is lethal. There is a strong relationship between VEGF expression and tumour aggressiveness, metastatic potential, a short time to relapse and consequently, elevated VEGF expression indicates poor prognosis in patients with cancer.
Angiogenesis is one of the most critical factors in tumour maintenance and progression. Malignant cells become capable of inducing phenotypic changes in endothelial cells as well as other cell types. Highly malignant astrocytomas may exhibit striking angiogenesis and markedly increased expression of VEGF. VEGF exerts multiple effects at the cellular level as a result of its ability to affect a complex integrated network of signalling pathways via three cognate receptors on surface of endothelial cells: VEGFR1 (Flt-1 ), VEGFR2 (KDR) and VEGFR3, and two non-tyrosine neuropilin receptors: NRP-1 and NRP-2. NRP-1 and NRP-2 are isoform-specific receptors of VEGF, which function as co-receptors with VEGFR2. A soluble truncated form of VEGFR1 (sFlt-1 ) binds VEGF as strongly as the full-length Flt-1 . sFlt-1 inhibits VEGF activity by sequestering it from signalling receptors and by forming non- signalling heterodimers with VEGFR2 (the so called decoy receptor). The VEGF receptors are primarily expressed in tumour vascular endothelium, and are absent in normal endothelial cells in surrounding tissue (Plate KH, Breier G, Weich HA, Mennel HD, Risau W. Vascular endothelial growth factor and glioma angiogenesis: coordinate induction of VEGF receptors, distribution of VEGF protein and possible in vivo regulatory mechanisms. Int J Cancer. 1994; 59:520-529).
The VEGFR2 antagonist Tivozanib (available from Aveo Pharmaceuticals Inc.) has demonstrated the overall median progression-free survival of patients in phase 2 trials to be 1 1 .8 months. The present invention demonstrates how targeting of VEGFR2 either directly or indirectly increases the sensitivity of cancer stem-like cells and/or progenitor cells to conventional cancer therapy and an increase in progression-free survival time is expected compared to conventional cancer therapy alone.
Summary of invention
The present invention demonstrates that VEGFR2 is expressed in cancer stem-like cells and progenitor cells and is required for their survival. By targeting VEGFR2 directly with antagonists to VEGFR2 or indirectly with antagonists to NRP-1 either alone or in combination, thereby inhibiting the activity of VEGFR2, it is possible to make the cancer stem-like cells and/or progenitor cells more susceptible to cancer therapy including radiation therapy. By targeting the cancer stem-like cells and/or progenitor cells either directly or indirectly through VEGFR2 the risk of recurrence of the cancer is reduced.
In one embodiment the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a vascular endothelial growth factor receptor 2 (VEGFR2) antagonist for treatment of a tumour selected from the group consisting of connective tissue tumours, tumours of the nervous system, hematologic tumours, lymphoid leukemias and/or myeloid leukemias in a subject in need thereof, wherein the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy.
In another embodiment the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a neuropilin-1 (NRP-1 ) antagonist for treatment of a tumour selected from the group consisting of connective tissue tumours, tumours of the nervous system, hematologic tumours, lymphoid leukemias and/or myeloid leukemias in a subject in need thereof, wherein the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy.
In a further aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a vascular endothelial growth factor receptor 2 (VEGFR2) antagonist for reducing the risk of recurrence of a
hyperproliferative disease in a subject in need thereof by inhibiting growth of cancer stem-like cells and/or progenitor cells. In one embodiment, the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy. In another embodiment, the pharmaceutical composition is prepared for administration to said subject prior to radiation therapy which would follow within days or weeks of administering said pharmaceutical composition.
In yet another aspect, the present invention provides a pharmaceutical composition comprising a therapeutically effective amount of a neuropilin-1 (NRP-1 ) antagonist for reducing the risk of recurrence of a hyperproliferative disease in a subject in need thereof by inhibiting growth of cancer stem-like cells. In one embodiment, the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy.
In addition the present invention enables identification of cancer stem-like cells and/or progenitor cells expressing VEGFR2 by identifying cells expressing VEGFR2. According, it is also an objective of the present invention to identify and optionally isolate cancer stem-like cells and/or progenitor cells. In one embodiment the identification of cancer stem-like cells and/or progenitor cells expressing VEGFR2 can be used for evaluating the presence and putative progression of a cancer disease. In one embodiment the present invention relates to a method for isolation of cancer stem-like cells and/or progenitor cells expressing VEGFR2 comprising contacting the population of cells with a molecule that specifically binds VEGFR2, and isolating cells that bind the molecule that specifically binds VEGFR2.
In still further aspects the present invention relates to a method of treating a tumour in a subject by inhibiting the cancer stem-like cells and/or progenitor cells expressing VEGFR2 by administering to the subject a therapeutically effective amount of a vascular endothelial growth factor receptor 2 (VEGFR2) antagonist and/or an antagonist of NRP-1 , and immediately thereafter subjecting the tumour in the subject to ionizing radiation therapy and optionally treatment with anti-VEGF antibodies. In one embodiment of the present invention, the method also relates to reducing the risk of recurrence of a tumour in a subject by inhibiting the cancer stem-like cells and/or progenitor cells.
It is also the objective of the present invention to provide a method of identifying a compound capable of inhibiting cancer stem-like cells and/or progenitor cells expressing VEGFR2 through binding to VEGFR2 said method comprising contacting VEGFR2 and/or a cell-line expressing VEGFR2 with a test compound, and detecting the autophosphorylation of VEGFR2 itself or/and activation of c-Raf/MAPK, P13K/Akt and/or PLCy1/PKC pathway, and selecting the compounds which inhibits an activation of said pathways. In one embodiment the VEGFR2 is operatively linked to a reporter gene and the activation of said reporter gene is measured. In yet another aspect of the present invention provides the use of the compound as identified herein for the manufacture of a medicament for the treatment of a cancer disease. In one embodiment the medicament is for reducing the risk of recurrence of a cancer disease.
Description of Drawings
Figure 1 : VEGFR2 was found to be expressed in astroglial cells on human glioblastoma specimens, associated with the so-called "vascular niche". Expression of VEGFR2 co-localizes with expression of the commonly used cancer stem-like cell marker - CD133. Presence of VEGFR2 positive cells was further confirmed by FACS in 6 other human specimens and the percentage of positive cells ranged from 0.8-35% (data not shown), where there was always presence of double-positive CD133/VEGFR2 cells. Figure 2: Co-staining with CD31 was done to exclude that the VEGFR2 positive glial cells are actually glioblastoma-derived tumour endothelial cells. Staining on frozen section from human specimen was performed. Co-staining of VEGFR2 with CD31 was done to determine the presence of tumour endothelial cell in our target fraction. As shown above, there is definitely fraction of VEGFR2 positive and CD31 negative cells.
Figure 3: Tumour sphere formation assay was performed on 3 different specimens. This assay is used to study self-renewing capacity (property of stem-like cells) of cells. We sorted one cell per well and were counting number of spheres (of more then 50 cells) formed by single cell. As shown above, CD133-VEGFR2 positive and single VEGFR2 positive show high tumour sphere forming capacity, compared to single CD133 positive and negative population.
Figure 4: Growth properties of VEGFR2 positive cells are significantly enhanced compared to their negative counterparts.
Figure 5: Shows unspecific binding of the two VEGFR2 selective inhibitors used at different concentrations, all below the concentration expected to target other tyrosine kinase receptors like EGFR, PDGFR. Unspecific binding in the present invention is binding of an inhibitor which is not specific to the VEGFR2 exclusively. In this figure two known VEGFR2 inhibitors is demonstrated to also bind VEGFR2 negative cells, hence the two inhibitors must have other targets and are not specific to VEGFR2.
Figure 6: IR-induced CD133-positive cancer stem-like cells' enrichment is abrogated by SU1498 in vitro. Astroglial tumour cells (A172 and U251 MG) were plated at 60% density. Next day, cells were treated with ionizing radiation of 8 Gy (dose rate 2.18 Gy/min by an X-ray generator, Pantak, Berkshire, United Kingdom; HF160; 150 kV; 15mA) after 1 hour pre-treatment with the inhibitor SU1498 or left untreated, and analyzed at indicated time after irradiation for the presence of CD133-positive cells using FACS Calibur (BD Biosciences). IR-induced enrichment of CD133-positive cancer stem-like cells was abrogated by simultaneus treatment with VEGFR2 tyrosine kinase inhibitor SU1498. Representative dot blots 72 hours post-treatment are shown in A. Corresponding graphs are shown in B. Assay was performed 3 times in duplicate (100000 cells per sample). Results are means ± s.d
Figure 7: A). Specimen #1966 did not show statistically significant difference in in vivo tumour formation assay when 100 cells injected.
B) Specimen #556 did show statistically significant difference in in vivo tumour formation assay when 100 cells injected.
C) Overall statistical analysis of both experiments shows significant difference in tumor formation, when 100 VEGFR2-positive and 100 VEGFR2-negative cells injected intracranially.
The label High denotes high level of VEGFR2, and the label Low denotes low level of VEGFR2.
Figure 8: Radiation does not efficiently kill VEGFR2 positive cells alone. Cells were freshly dissociated (from mouse xenograft #556 (A and B) and from patient #0607 (C and D)), recovered over night, plated and treated for 2 hours with the VEGFR2 antagonist SU1498 or left untreated. After this the cells were irradiated with 8 Gy or left without radiation. 24 hours (A and C) and 72 hours later (B and D), surviving cells were analyzed by FACS for presence of CD133, VEGFR2 and VEGFR2-CD133 positive cells. VEGFR2+ means small sub-population of cells which attach and resembles endothelia cells.
Fig. 9 VEGFR2 expression in gliomas VEGFR2 is expressed by human glioma initiating cells (GICs). (A) FACS analysis of freshly dissociated human glioma specimens (#1 -#17) shows VEGFR2 to be preferentially expressed in a fraction of CD133-positive cells (**p = 0.0017; mean positivity 10.5%) in comparison to their negative counterparts (mean positivity 3.252%). (A). Interestingly, we observed that glioma cells not only express membrane VEGFR2, but a substantial fraction ranging from 12 to 27% cells have VEGFR2 internalized as measured by FACS analysis of extracellular versus total VEGFR2 staining (C) and can be detected directly by immunohistochemistry staining on paraffin-embedded sections from corresponding glioma specimens (* labels membrane localization, Δ labels internalized VEGFR2) (B).
Fig.10 VEGFR2-positive cells recapitulate patient phenotype when injected in immunocompromised rodent host.
After subcutaneous implantation of VEGFR2-positive cells, these form tumors, with distinct areas of VEGFR2 expression reminding vessel-like structures (A). VEGFR2 is autophosphorylated (B), which indicates constitutive activation of VEGFR2 receptor in these cells. Interestingly and in agreement with recently published literature (Nature, 2010), when injected into mouse brain parenchyma, GFP-labeled VEGFR2-positive cells contribute to vessel formation (C).
Fig.11 VEGFR2 expression in gliomas accounts for growth advantage and increased VEGF secretion
(A) As shown by histogram plot from FACS analysis, VEGFR2-low (L; surface negative), VEGFR2-medium (M; surface negative) and VEGFR2-high (H; surface positive) cells were sorted out to confirm the presence of internalized VEGFR2 by Western blot analysis (see on the right). As shown, VEGFR2-low cells do not have internalized VEGFR2, whereas it could be detected in VEGFR2-medium cells. Interestingly, neuropilinl (NRP1 ) protein levels (VEGFR2 co-factor contributing to enhanced VEGFR2/VEGF signaling) were gradually increased with increased VEGFR2 expression indicating importance of neuropilins in VEGFR2 signaling. (B) VEGFR2- high cells have significantly high self-renewing capacities in sphere formation assay and enhanced viability in vitro (C). VEGFR2-high cells exhibit enhanced secretion of VEGF ligand when compared to VEGFR2-negative cells (D). Most common antiangiogenic drug AVAstin (bevacizumab) efficiently binds and blocks VEGF ligand as expected and SU1498 tyrosine kinase inhibitor (VEGFR2 specific), decreases VEGF secretion indication of autocrine loop signaling (E).
Fig.12 VEGFR2 is stabilized by NRP1 and remains constitutively active in endosomal compartment
(A) Co-immunoprecipitation was performed to investigate the interaction of VEGFR2 with its putative co-receptor Neuropilin-1 (NRP1 ). Cells from freshly dissociated specimen #556 were left to recover overnight, pre-treated with SU1498 (10 mM) or Bevacizumab (0.5 mg/ml) followed by 10 minute exposure to recombinant VEGF165 or left untreated and then submitted to immunoprecipitation with rabbit monoclonal antibody against VEGFR2. Immunoprecipitates were submitted to Western blot analysis for the presence of Neuropilin-1 . Stimulation with exogenous VEGF ligand increased interaction of VEGFR2 and NRP1 . In contrast to ligand sequestration by Bevacizumab, which lead to abrogation of this interaction, the pre-treatment with tyrosine kinase inhibitor SU1498 did not exhibit any significant effect on VEGFR2- NRP1 complex formation. (B) shRNA knock-down of NRP1 leads to destabilization and decreased levels of VEGFR2. (C) Membrane labeling with biotin was performed to distinguish between cytosolic (biotin non-labeled) and membrane integrated (biotin- labeled) VEGFR2 fraction in order to investigated putative autophosphorylation of the receptor in intracellular compartment. As shown here, bioton labeling revealed that substantial fraction of VEGFR2 is constitutively active in cytosolic compartment, whereas very little of the protein is presented on the cell surface. (D) Confocal microscopy imaging has shown VEGFR2 granular pattern of expression and these granule to be co-localized with EEA1 (a marker of early endosomes) and not to Rab4b (marker of recycling endosomes). (E) Membrane fractionation of cells with heterogenous VEGFR2 expression (parental non-sorted cells) has shown VEGFR2 to be present primarily in cytosolic fraction (co-segregated with early endosomal antigen 1 (EEA1 ) as indicated by confocal microscopy results in (D).
Fig.13 Inhibition of VEGFR2 tyrosine kinase, not VEGF ligand blockage is crucial for abrogation of VEGFR2 autocrine signaling Abrogation of VEGFR2 tyrosine kinase activity leads to apoptosis, whereas VEGF ligand sequestration by blocking antibody (Bevacizumab, 0.5 mg/ml) has no significant effect on their survival. Freshly dissociated xenografted #556 cells were left to recover overnight and next day treated as follows: 10 mM SU1498 (A) or Bevacizumab (B, 0.5 mg/ml) pre-treated or not treated for 2 hours and further irradiated or sham-irradiated (8 Gy). Cells were collected 12 and 24 hours post-IR treatment and analyzed using FACS for VEGFR2-AlexaFluor647/AnnexinV-AlexaFluor488/7-AAD co-staining. For analysis, cells were sub-gated using FlowJo software as VEGFR2high and VEGFR2|0W sub- populations. Next, each sub-population was separately analyzed for the presence of apoptotic (AnnexinV/7-AAD co-staining) and viable cell fractions. Apoptosis induction was observed 12 and 24 hours post single SU1498- as well as combined SU1498/IR treatment compared to untreated control. Error bars indicate s.d. (n=3). No significant effect was observed in case of single Bevacizumab treatment, indicating that VEGF ligand sequestration is not sufficient to abrogate the function of pro-survival VEGFR2 signaling. Moreover, VEGFR2 tyrosine kinase inhibitor SU1498 (alone, or followed by IR) significantly inhibited cell growth followed over 7-day period (C). This effect was lacking when Bevacizumab was used. (D) In vitro pre-treatment of VEGFR2 tyrosine kinase activity in VEGFR2hlQh cells decreases tumorigenesis in vivo (p=0.0078). Interestingly, pre-treatment with VEGF blocking antibody Bevacizumab did not lead to significant impairment of tumor formation.
Fig.14 Lentiviral mediated shRNA targeting of VEGFR2 results in enhanced apoptosis, decreased viability and impaired tumor formation
(A) In vivo limiting dilution assay shows significant difference in tumor formation capacities between VEGFR2high and VEGFR2|0W cell population. Kaplan-Meier survival curves demonstrate decreased survival when cells high in VEGFR2 expression are transplanted into the right frontal lobe of immunocompromised mice (p<0.01 for 100 #556 cells) tested with log-rank analysis of survival curves. Injection of 1000, 5000 or 10000 cells did not show any growth advantages and increase in tumor formation of VEGFR2high cells. (B) shRNA knockdown of VEGFR2 (sh1 , sh2 targeting VEGFR2 and control non-targeting shRNAs were used) intracranial xenograft model in mice. VEGFR2high cells were FACS-sorted using MoFlow XDP and after over-night recover infected with lentiviral shRNA particales (equal titers for control non-targeting and two independent VEGFR2-targeting shRNA particles was used). Second day post-infection, cells were selected with puromycin for 2 additional days. Next, cells were counted and 10 000 cells were injected into the right frontal lobe of immunocompromised mice (5 mice each group). Graph representing Kaplan-Meier survival curves demonstrate statistically significant increased mouse survival when VEGFR2 expression was successfully targeted with shRNA (p=0.0067 with log-rank analysis of survival curves for sh2). (C) VEGFR2 knockdown (sh1 and sh2) led to increase in cell death as evaluated by relative caspase 3/7 activity compared to non-targeting shRNA (shNT). Increased induction of apoptosis was accompanied by decreased cell viability over a period of 7 days (D) as assessed by CellTiter Glo Kit (Promega). Two independent shRNA targeting VEGFR2 shRNA efficiently inhibited cell viability compared to non- targeting control shRNA. Error bars indicate s.d. (n=3). Fig.15 VEGFR2 represents a protein with very short half-life and is induced by ionizing radiation.
(A) VEGFR2high cells (#556) were sorted using FACS sorting, left recover overnight in GF free medium and then treated with cyclohexamide for 30 min., 1 hour, 3 hours, 6 hours or left untreated and analyzed for VEGFR2 protein stability using semiquantitative Western blot analysis. Photoshop software was employed to calculate raw and relative intensities of corresponding bands. Further, the half-life of 42 minutes was estimated. Graph with exponential decay curve used for half-life estimation is shown (y- axis represents normalized relative intensity; x-axis represents Time in minutes). (B) MG132 (proteosomal inhibitor) was used in VEGFR2high cells to investigate further protein stabilization/accumulation after proteasomal inhibition. Already after 10 minutes post treatment, we observed increase in VEGFR2 protein levels, indicating of strong impact of protesomal degration pathway on VEGFR2 protein turnover. (C) To analyze to effect of ionizing radiation (IR) on VEGFR2-protein level, we treated VEGFR2- expressing cells with the dose of 8 Gy of IR and used Western blot analysis to check for total and phosphorylated VEGFR2 levels. As shown here, radiation induces total and phospho-VEGFR2 expression and pro-survival VEGFR2 signaling over 48 hours post radiation. At 72 hours pos radiation, VEGFR2 levels returned to baseline expression a compared to control. Detailed description of the invention
The term "treating" and "treatment", as used herein, unless otherwise indicated, refers to reversing, alleviating, inhibiting the process of, the disease, disorder or condition to which such term applies, or one or more symptoms of such disease, disorder or condition and includes the administration of an antagonist to alleviate the symptoms or the complications, or eliminate the disease, condition, or disorder. Preferably treatment is curative or ameliorating. In one embodiment of the invention the antagonist can be administered to prevent the disease or the onset of the symptoms or the complications of the disease.
The term "anti-neoplastic compound" or "anti-neoplastic agent" as used herein, unless otherwise indicated, denotes a cytotoxic compound which can be used as cancer chemotherapy to inhibit and combat cancer cells and tumours. "Anti-neoplastic compound" or "anti-neoplastic agent" can also be used to denote immune stimulating agents, antibodies, and cancer vaccines as described in the section "combination treatment" herein below.
The term "proliferative disease" or "hyperproliferative disease" as used herein, unless otherwise indicated, denotes a disease in which tissue, such as cells, grow or spread at a rapid abnormal rate, such as for example in cancer.
The term "therapeutically effective amount" as used herein, unless otherwise indicated, denotes an amount of a molecule which exerts a relevant therapeutically effect in a subject to which the molecule is administered.
The term "immediately prior to radiation therapy" as used herein, unless otherwise indicated, denotes an administration within 5 days prior to initiation of radiation therapy, more preferably within 4 days, such as within 3 days, more preferably within 2 days, such as within 1 day, more preferably within 20 hours, such as within 10 hours, more preferably within 5 hours, more preferably within 4.5 hours, such as within 4 hours, more preferably within 3.5 hours, such as within 3 hours, more preferably within 2.5 hours, such as within 2 hours, more preferably within 1 .5 hours, such as within 1 hour, more preferably within 0.5 hour, such as within 15 minutes, more preferably within 10 minutes, such as within 5 minutes prior to radiation therapy. The term "before radiation therapy" as used herein, unless otherwise indicated, denotes an administration within 3 weeks prior to initiation of radiation therapy, more preferably with 2 weeks, such as within 1 week, more preferably within 6 days. The term "before treatment with anti-VEGF antibodies" as used herein, unless otherwise indicated, denotes an administration within 5 days prior to administration of anti-VEGF antibodies, more preferably within 4 days, such as within 3 days, more preferably within 2 days, such as within 1 day prior to administration of anti-VEGF antibodies.
Biological activity refers to the biologically useful effects of a molecule on a specific cell. As used herein "a biologically active compound" is one which when administered to a subject exerts its effect on a target cell. The term "radiosensitizing drug" as used herein, unless otherwise indicated, denotes a drug which increases the sensitivity of targeted tumour cells towards radiotherapy.
The term "subject" as used herein, unless otherwise indicated, denotes a living animal. In preferred embodiments the subject is a mammal, including humans and non-human mammals such as dogs, cats, pigs, cows, sheep, goats, horses, rats and mice. In the most preferred embodiment, the subject is a human.
The term "reducing the risk of recurrence" as used herein, unless otherwise indicated, denotes a reduced risk of recurrent cancer as described in the section "recurrent cancer", so that at the most 90% of the subjects demonstrates a recurrent cancer, more preferably at the most 80%, such as at the most 70%, more preferably at the most 60%, such as at the most 50%, more preferably at the most 40%, such as at the most 30%, more preferably at the most 20%, such as at the most 10%, more preferably at the most 5%, such as at the most 4%, more preferably at the most 3%, such as at the most 2%, most preferably at the most 1 %. The method of the present invention is especially useful for reducing the risk of recurrence if the VEGFR2 antagonist of the present invention is combined with radiation therapy as described in the section "radiation therapy" The term "cancer stem-like cell" as used herein, unless otherwise indicated, denotes cells with proliferative potential, said cells expressing a variety of different markers, such as CD133, VEGFR2, and integrin Alpha-6. The term "progenitor cell" as used herein, unless otherwise indicated, denotes a biological cell, which has a tendency to differentiate into a specific cell type. The progenitor cell is more specific than a stem cell and may be directed to differentiate into its "target" cell. The most important difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, whereas progenitor cells can only divide a limited number of times.
Recurrent cancer
The term "recurrence" and "recurrent hyperproliferative disease" as used herein, unless otherwise indicated, denotes that a hyperproliferative disease which has been subject to treatment is reoccurring in the same patient. Preferably, a recurrent hyperproliferative disease is a cancer, which has come back in a patient who was thought to be cancer-free or in remission after treatment.
The hyperproliferative disease, such as cancer can come back:
- in the same organ or tissues where it started or in nearby tissues - this is called local recurrence
- in lymph nodes near the original cancer - this is called regional recurrence
- in other organs or tissues - this is called distant recurrence Distant recurrence is also called metastatic recurrence. For example, the hyperproliferative disease might recur in distant parts of body, such as in bones, the liver, or the lungs. This may happen because some cancer cells, in particular cancer stem-like cells, have broken off from the original tumour, travelled through the body, and are establishing tumours elsewhere.
When the patient is evaluated to be free of tumours using conventional detection methods employed in the field of oncology, a cancer is considered to be in remission (the temporary or permanent absence of disease). A recurrence is defined as the return of cancer after a period of remission. Typically cancer recurs because undetected surviving cancer cells in particular cancer stem-like cells frequently remain in the body after treatment. Over time, these undetected cells can multiply and grow large enough to be recognized and diagnosed using the conventional methods of diagnosis employed in the field of oncology today. Depending on the type of cancer, this can happen in weeks, months, or even many years after the primary (original) cancer was treated.
The likelihood that a cancer will come back depends on the type of the primary cancer. When and where a cancer will recur also varies. Most cancers have a predictable pattern of recurrence. Brain tumours and especially astrocytoma is a cancer type with a high rate of recurrence. In one embodiment of the present invention the risk of recurrence, as defined above, is reduced.
Cancer stem-like cells and/or progenitor cells
The present invention relates to targeting cancer stem-like cells and/or progenitor cells expressing VEGFR2. Furthermore the present invention describes antagonists of VEGFR2. In particular the invention describes methods for eliminating cancer stem-like cells by inhibiting VEGFR2 signalling through antagonist blocking of the receptor rendering the cancer stem-like cells and/or progenitor cells sensitive to conventional cancer treatment such as radiation therapy. The inhibition of VEGFR2 signalling may be directly or indirectly. In preferred embodiment the inhibition is however direct. Indirect inhibition of VEGFR2 signalling is preferably through NRP-1 receptor. Inhibition of VEGFR2 may in one embodiment be accomplished by a combination of VEGFR2 inhibitors and NRP-1 inhibitors. Also the invention describes methods for detecting cancer stem-like cells and/or progenitor cells by identifying the presence of VEGFR2 expressing cells in a cell sample, said cell sample preferably obtained from a patient suffering from a hyperproliferative disease. The detection of cancer stem-like cells and/or progenitor cells is in one embodiment performed by identifying the presence of a variety of other markers, such as CD133 preferably in combination with the identification of the presence of VEGFR2. The presence of VEGFR2 may in one embodiment be identified using antibodies directed against VEGFR2, such as antibodies describes in the section "Antagonists"
Cancer stem-like cells and/or progenitor cells are cells with proliferative potential, said cells expressing a variety of different markers, such as CD133, VEGFR2, and integrin Alpha-6. Cancer stem-like cells and/or progenitor cells expressing a high level of VEGFR2 demonstrates the capability of tumour formation when only 100 cells are injected in a subject (example 1 and figure 7).
Recently, a small population of cancer stem-like cells (CSCs) has been identified in adult and paediatric brain tumours, as well as in established cell lines. These cells express a number of neural stem-cell (NSC) markers including CD133, which may be used for enrichment of stem-like cells using fluorescence activated cell sorting (FACS) and/or magnetic bead separation. The human CD133 antigen, also known as AC133, was originally identified as a marker of haematopoietic stem-like cells. Besides expressing the CD133 antigen, cancer stem-like cells express mRNAs for several additional recognized NSC markers, including bmi-1 , Sox2 and musashi-1 (Hemmati HD, Nakano I, Lazareff JA, Masterman-Smith M, Geschwind DH, Bronner-Fraser M, Kornblum HI. Cancerous stem-like cells can arise from pediatric brain tumors. Proc Natl Acad Sci U S A. 2003; 100: 15178-83). Some cancer stem-like cells have been found to CD133-negative, and hence CD133 is not necessarily the best marker of cancer stem-like cells. The present invention demonstrated that VEGFR2 is expressed also in CD133-negative cancer stem-like cells.
Cancer stem-like cells may be cultivated in vitro e.g. brain tumour-derived cancer stem- like cells (BTCSCs) may be cultured according to the following criteria: i) Expression of NSC markers CD133 and nestin (a marker of glial cells); ii) Generation of spheres morphologically indistinguishable from neurospheres; iii) Self renewal and proliferation; and iv) Production of differentiated progeny in vitro or recapitulation of the parental tumour mass growth when implanted into immunodeficient animals. As few as one hundred CD133-positive human cells were sufficient to form brain tumours as xenografts in NOD-SCID mice (Singh SK, Hawkins C, Clarke ID, Squire JA, Bayani J, Hide T, Henkelman RM, Cusimano MD, Dirks PB. Identification of human brain tumour initiating cells. Nature. 2004; 432: 396-401 ). Importantly, the CD133-positive BTCSCs also exhibit high resistance to current cytotoxic drugs and radiotherapy, in contrast to their CD133-negative counterparts (Liu G, Yuan X, Zeng Z, Tunici P, Ng H, Abdulkadir IR, Lu L, Irvin D, Black KL, Yu JS. Analysis of gene expression and chemoresistance of CD133+ cancer stem-like cells in glioblastomas. Mol Cancer. 2006; 5: 67, Bao S, Wu Q, McLendon RE, Hao Y, Shi Q, Hjelmeland AB, Dewhirst MW, Bigner DD, Rich JN. Glioma stem cells promote radioresistance by preferential activation of the DNA damage response. Nature. 2006; 444: 756-60). The finding that cancer stem-like cells are present within the tumour mass could explain the recurrence of cancer and targeting of the cancer stem-like cells are the main objective of the present invention. Figure 3 and example 1 of the present invention describes one possible way of examing the self renewing capacity of cells, hence evaluating the cancer stem-like cell potential.
The present invention demonstrates that VEGFR2 is enriched in cancer stem-like cells in brain tumours. VEGFR2 positive cells demonstrate self-renewing capacity (property of stem-like cells) of cells. As shown in figure 3 of the present invention CD133- VEGFR2 positive and single VEGFR2 positive cells show high tumour sphere forming capacity (a measurement of self-renewing capacity), compared to single CD133 positive and negative population.
As demonstrated in figure 4 growth properties of VEGFR2 positive cells are significantly enhanced compared to their negative counterparts.
The present invention demonstrates that by targeting VEGFR2 expressing cancer stem-like cells, said cancer stem-like cells being CD133-positive, the sensitivity of the cancer stem-like cells to conventional cancer treatment is increased. In a preferred embodiment the conventional cancer treatment is radiotherapy.
VEGFR2
The present invention relates to VEGFR2 antagonists, said antagonist described in the section "antagonists". The antagonist of the present invention may be used for inhibiting the signalling through the wild-type VEGFR2 (SEQ ID NO:1 ). The VEGRF2 is involved in angiogenesis, differentiation and host-virus interactions. VEGFR2 is also denoted KDR/Flk-1 and is a 152 kDa single-pass type 1 membrane protein. It belongs to the protein kinase superfamily and the Tyr protein kinase family. VEGFR2 contains seven Ig-like (Immunoglobulin-like) C2-type domains located in the extracellular part and one protein kinase domain located in the intracellular part. The protein kinase domain is located on amino acid 834-1 162 of SEQ ID NO:1 . Several natural variants of this receptor exists as described in UniProt (SEQ ID NO: 2-16). The present invention also relates to antagonists of functional homologues of VEGFR2. In the present invention functional homologues is VEGFR2 variants with the same function as wildtype VEGFR2, said functional homologous having changes in the amino acid sequence as compared to SEQ ID NO:1 . Common to all functional homologous of the present invention is the conservation of Arg82, Lys84 and His86 of VEGFR2 (numbering according to SEQ ID NO:1 ). In one embodiment the present invention relates to functional homologues of VEGFR2 antagonists. Within the scope of the present invention a functional homologue is a polypeptide that exhibits some sequence identity with a known VEGFR2 antagonist as the antagonist described in the section "antagonists", sharing at least 50%, preferably at least 60%, more preferably at least 75%, even more preferably at least 80%, yet more preferably at least 85%, even more preferably at least 90%, yet even more preferably at least 95%, and most preferably at least 99% identity with known VEGFR2 antagonists.
Sequence identity can be calculated using a number of well-known algorithms and applying a number of different gap penalties. In relation to the present invention the sequence identity should be calculated relative to the full-length sequence of a known VEGFR2 antagonist. Any sequence alignment tool, such as but not limited to FASTA, BLAST, or LALIGN may be used for searching homologues and calculating sequence identity. Moreover, when appropriate any commonly known substitution matrix, such as but not limited to PAM, BLOSSUM or PSSM matrices, may be applied with the search algorithm. VEGFR2 is almost exclusively expressed in endothelial cells, however the present invention has revealed that VEGFR2 also is present in cancer stem-like cells and/or progenitor cells. This expression of VEGFR2 in cancer stem-like cells and/or progenitor cells has been found to be present in both CD133 negative cancer cells as well as CD133 positive cancer stem-like cells. Cancer stem-like cells and/or progenitor cells expressing a high level of VEGFR2 demonstrate the capability of tumour formation when only 100 cells are injected in a subject (example 1 and figure 7).
The present invention demonstrates that by targeting VEGFR2 expressing cancer stem-like cells and/or progenitor cells, the sensitivity of the cancer stem-like cells and/or progenitor cells to conventional cancer treatment is increased. In a preferred embodiment the conventional cancer treatment is radiotherapy.
The present invention demonstrates that by targeting VEGFR2 with a VEGFR2 antagonist prior to radiation the effects of radiation-induced VEGF secretion is minimised and the efficiency of the radiation treatment hence increased by targeting the unique features of the candidate cancer stem-like cells and/or progenitor cells. In general terms, such a combined strategy may include available or future VEGFR2 antagonists or antibodies to inhibit VEGFR2 as described in the section "antagonist", combined with standard DNA damaging treatment modalities such as ionization radiation therapy and alkylating drugs, complemented by selective inhibitors of checkpoint signalling or DNA repair to counteract the mechanisms underlying tumour resistance to such treatments. In one embodiment the combined strategy for treatment may be made as multiple treatments during longer periods of time.
Method of detecting a cancer stem-like
In one embodiment the present invention relates to a method of detecting a cancer stem-like cell and/or progenitor cells expressing VEGFR2 in a population of isolated cells, said cells preferably being isolated from a tumour. As described in the section "cancer stem-like cells" and "VEGFR2" cancer stem-like cells and/or progenitor cells expressing VEGFR2 are important for the treatment resistance of tumours to conventional cancer therapy. Cancer stem-like cells and/or progenitor cells of the present invention holds self renewal potential and proliferative potential and said cancer stem-like cells and/or progenitor cells expresses VEGFR2, preferably without expression of CD31 , CD31 being a marker for endothelial cells. The present invention demonstrates that by targeting VEGFR2 expressing cancer stem-like cells and/or progenitor cells, said cancer stem-like cells and/or progenitor cells being CD133- positive, the sensitivity of the cancer stem-like cells and/or progenitor cells to conventional cancer treatment is increased. In a preferred embodiment the conventional cancer treatment is radiotherapy. In another preferred embodiment the conventional cancer treatment is administration of anti-VEGF antibodies. By detecting the presence of cancer stem-like cells and/or progenitor cells expressing VEGFR2 in a population of isolated cells, said cells preferably being isolated from a tumour, the present invention makes it possible to make a diagnosis, preferably before the tumour is detected using conventional methods of detecting the presence of tumours in a subject. It also makes it possible to determine the potential of a tumour of reoccurrence, because the presence of cancer stem-like cells and/or progenitor cells expressing VEGFR2 in a tumour indicates a potential for reoccurrence.
Said tumour is in one embodiment selected from the group consisting of connective tissue tumours, tumours of the nervous system, hematologic tumours, lymphoid leukemias and/or myeloid leukemias. Preferably said tumour is a tumour of the nervous system, more preferably a tumour selected from the group consisting of glioma, glioblastoma, glioblastoma multiforme, astrocytoma, oligodendroglioma, ependymomas, medulloblastoma, meningioma, neurinoma and Schwannoma, even more preferably the tumour is a glioblastoma or glioblastoma multiforme.
The steps of the method according to the invention could in one embodiment be performed under conditions suitable for maintaining the cell morphology i.e. the cytoplasm and nucleus of the cells.
In an embodiment the invention relates to a method of detecting a cancer stem-like cell and/or progenitor cells expressing VEGFR2 in a population of isolated cells comprising the following steps:
- contacting the population of cells with a molecule that specifically binds VEGFR2, and - detecting cells that bind the molecule that specifically binds VEGFR2, and
- optionally isolating cells that have been detected and
- distinguishing any endothelial cells from the cancer stem-like cells and/or progenitor cells. In preferred embodiments of the invention the molecule that specifically binds VEGFR2 is labelled by a detectable label. The label may be any of the labels described herein below in the section "Labelling". However, the molecule that specifically binds VEGFR2 may also be detected using another molecule that specifically binds said molecule that specifically binds VEGFR2, wherein said other molecule is labelled using a detectable label.
Depending on the nature of the detectable label it may be detected using conventional methods. If the detectable label is a fluorescent label it may be detected using e.g. a fluorescence microscope, plate reader or a FACS.
In the present invention distinguishing said endothelial cells may be done by contacting said sample with a molecule specifically binding endothelial cells. Said molecule specifically binding endothelial cells may in a preferred embodiment be labelled by a detectable label, more preferably different detectable label from the label of the molecule that specifically binds VEGFR2. Alternatively, the molecule that specifically binds endothelial cells may be detected by another molecule specifically binding thereto. Said other molecule may then be labelled by a detectable label. Preferably, the sample may then be subjected to a separation step, e.g. by FACS analysis. In one embodiment the sample is a tissue section mounted on a glass slide and distinguishing said endothelial cells may be done by evaluating the tissue section contacted with said molecules, said molecules preferably being labelled, for the presence of the specific molecules. In one embodiment the molecule that specifically binds VEGFR2 is an antagonist as described in the section "antagonist" and the molecule that specifically binds endothelial cells is an antibody selected from the group consisting of antibodies specifically binding one or more selected from the group consisting of CD31 , CD34, Tusc5, 7B4, BNH9/BNF13, CD105, CD146, D2-40, EN4 and PAL-E. Preferably said antibody specifically binds CD31 .
The endothelial cells may be removed from the sample by contacting said sample with a molecule specifically binding endothelial cells, said molecule such as an antibody specific for the endothelial cells. Said molecule, such as an antibody may preferably be coupled to a solid support e.g. a column or another suitable material. In one embodiment said antibody is selected from the group consisting of antibodies specifically binding CD31 , CD34, Tusc5, 7B4, BNH9/BNF13, CD105, CD146, D2-40, EN4 and PAL-E. Preferably said antibody specifically binds CD31 . Any cells not binding the specific molecule can be removed by simple washing and subjected to further analysis according to the present invention.
According to the method of the present invention the cancer stem-like cell and/or progenitor cells expressing VEGFR2 may be detected using various suitable techniques. In order to detect the cancer stem-like cells and/or progenitor cells expressing VEGFR2, the cancer stem-like cells and/or progenitor cells expressing VEGFR2 are selectively labelled. In one embodiment of the present invention the selective labelling of the cancer stem-like cells and/or progenitor cells expressing VEGFR2 is done by the specific recognition of the cancer stem-like cells and/or progenitor cells expressing VEGFR2 by a specific molecule, said molecule preferably being an antibody, said antibody preferably being a VEGFR2 specific antibody.
In one embodiment detection of cancer stem-like cells and/or progenitor cells expressing VEGFR2 is performed by co-labelling the cell sample with a molecule that specifically binds VEGFR2, wherein said molecule may be any of the VEGFR2 antagonists being described in the section "antagonists", and one or more other cancer stem-like cell and/or progenitor cells expressing VEGFR2 marker. In another embodiment the detection of cancer stem-like cells and/or progenitor cells expressing VEGFR2 is performed by co-labelling the cell sample with VEGFR2 specific molecule and CD133 and/or integrin Alpha-6. In a preferred embodiment the detection of cancer stem-like cells expressing VEGFR2 is performed by co-labelling the cell sample with VEGFR2 specific molecule and CD133. In one embodiment detection of cancer stem-like cells and/or progenitor cells expressing VEGFR2 is performed so as to detect both membrane expressed VEGFR2 and internalized VEGFR2. This is done by dual labelling. Firstly, dissociated tumour cells (e.g. paraffin sections) are subjected to primary-conjugated VEGFR2 antibody which would detect the membrane expressed VEGFR2. After washing out non-bound antibody, cells are fixed, permeabilized and co-labelled with the same antibody to achieve the total staining for VEGFR2. Only a small fraction of total VEGFR2 is present on the surface of cancer stem-like cells and/or progenitor cells as demonstrated in figure 9. In the present invention detection of cancer stem-like cells and/or progenitor cells expressing VEGFR2 may in one embodiment be used for diagnosis of patients suffering from a hyperproliferative disease, preferably a cancer. By detecting the presence of cancer stem-like cells and/or progenitor cells expressing VEGFR2 it is possible to diagnose subjects suffering from a hyperproliferative disease and the amount of VEGFR2 expressing cancer stem-like cells and/or progenitor cells expressing VEGFR2 could in one embodiment be used as an indication of the prognosis for said hyperproliferative disease in said subject. The detection of a high number of detected cancer stem-like cells and/or progenitor cells expressing VEGFR2 increases the severity of the hyperproliferative disease and the prognosis for the subject is hence not good. The more cancer stem-like cells and/or progenitor cells expressing VEGFR2 present in a sample from a subject suffering from a hyperproliferative disease, the worse the prognosis.
In one embodiment of the present invention the detection of cancer stem-like cells and/or progenitor cells expressing VEGFR2 may be used to identify patients sensitive to treatment with VEGFR2 and/or NRP-1 antagonist as described herein below in the section "Method of treatment".
In a preferred embodiment the detection of cancer stem-like cells and/or progenitor cells may be used for identification of patients suffering from glioblastoma multiforme (GBM). Patients suffering from GBM have a very bad prognosis and early identification of such patients would be highly beneficial. The patients identified to be suffering from GBM may be treated as described herein below in the section "Method of treatment". Identification of VEGFR2 antagonist
In one embodiment of the invention VEGFR2 can be used for identifying a compound capable of inhibiting cancer stem-like cells and/or progenitor cells expressing VEGFR2 through binding to VEGFR2, said method comprising contacting VEGFR2 with a test compound and detecting autophosphorylation of VEGFR2 and/or activation of c- Raf/MAPK, P13K/Akt and/or PLCyl/PKC pathway and selecting the compounds which inhibits the activation of said pathways. In one embodiment VEGFR2 are expressed on cells or cell lines and said cells or cell lines are then contacted with the test compound and autophosphorylation of VEGFR2 and/or activation of c- Raf/MAPK, P13K/Akt and/or PLCyl/PKC pathway is detected and compounds which inhibits the activation of said pathways are selected. The cell or cell line may be a primary cell, a primary cell culture or an established cell line. Thus, the cell line may for example be selected form the group consisting of U1 18MG, U87MG, T98G, A172, Gli-06, SF126, U373MG and U251 MG. The cells may for example be selected from the group consisting of primary cells from Glioblastoma Multiforme, primary cells from patients diagnosed with cancer, primary cell lines and primary xenograft cells.
In another embodiment the method of identifying a compound capable of inhibiting cancer stem-like cells and/or progenitor cells through binding to VEGFR2, said method comprising VEGFR2 linked to a reporter gene, said reporter-linked VEGFR2 is contacted with the test compound and the activation of said reporter gene is detected. The compounds, which inhibits the activation of said reporter gene is selected. A reporter gene of the present invention is a gene construct with a promoter operatively linked to VEGFR2 said promoter initiating the expression of an enzyme or other compound (reporter gene product) which induce visually identifiable characteristics. In one embodiment such a reporter gene product could be fluorescent and luminescent proteins, such as for example Green Fluorescent Protein (GFP) and luciferase, respectively. The activation of VEGFR2 is then measured by the activation of the reporter gene and hence the formation of gene product visualized in one embodiment as fluorescent protein, which can be observed using standard methods known in the art to observe fluorescence.
In one embodiment the detection of activation is done by Western blot analysis.
In a preferred embodiment the VEGFR2 antagonist identified is able to pass the cellular membrane of cancer stem-like cells and/or progenitor cells as well as the blood-brain barrier. Antagonists
The present invention relates to the field of diagnosis and treatment of hyperproliferative diseases, such as cancer. In particular, the invention relates to methods of identifying and killing cancer stem-like cells and/or progenitor cells expressing VEGFR2 using compounds capable of associating with VEGFR2 and in particular VEGFR2 antagonists and/or NRP-1 antagonists. Furthermore, the invention relates to identification of compounds useful for identifying and killing cancer stem-like cells and/or progenitor cells, said compounds being VEGFR2 antagonists.
An antagonist of the present invention could also be directed at the native ligand for VEGFR2 (e.g. VEGF), however it is preferred that the antagonist of the present invention is directed at the receptor VEGFR2 as receptor specific antagonists are more effective and independent of receptor activation. The VEGFR2 also generates VEGF upon activation and targeting VEGF would hence require a higher amount of antagonist to achieve the same IC50 value (described below in this section "antagonists"). In the present invention it is preferred that the antagonist is targeting VEGFR2.
An antagonist of the present invention could also in another embodiment be directed at neuropilin-1 (NRP-1 ). NRP-1 is a isoform-specific receptor of VEGF, which function as co-receptor with VEGFR2 (see figure 12). Inhibiting NRP-1 would indirectly inhibit the activity of VEGFR2. In the present invention a NRP-1 antagonist may be a small molecule, peptides and/or proteins. In the present invention an antagonist targeting NRP-1 may be administered alone or in combination with antagonist targeting VEGFR2 directly. In a preferred embodiment the NRP-1 antagonist and the VEGFR2 antagonist is administered in combination. A VEGFR2 antagonist according to the present invention is a type of VEGFR2 ligand or drug that does not provoke a biological response itself upon binding to VEGFR2, but blocks or dampens agonist-mediated responses. In the present invention, antagonists have affinity but lower efficacy for VEGFR2, preferably the antagonist of the present invention have low efficacy for VEGFR2, more preferably no efficacy for VEGFR2. In the present invention a molecule with efficacy to the VEGFR2 is a molecule which can activate the downstream pathways linked to said receptor. Said pathways are the c- Raf/MAPK, P13K/Akt and/or PLCyl/PKC pathway. Binding of the VEGFR2 antagonist will disrupt the interaction and inhibit the function of an agonist (such as VEGF) or inverse agonist at receptors. Antagonists may mediate their effects by binding to the active site or to allosteric sites on receptors, or they may interact at unique binding sites not normally involved in the biological regulation of the receptor's activity. Antagonist activity may be reversible or irreversible depending on the longevity of the antagonist-receptor complex, which, in turn, depends on the nature of antagonist receptor binding. The majority of drug antagonists achieve their potency by competing with endogenous ligands or substrates at structurally-defined binding sites on receptors.
In a preferred embodiment the antagonists of the present invention should be able to pass the cellular membranes of cancer stem-like cells and/or progenitor cells as well as the blood-brain barrier of a human subject.
The potency of an antagonist is usually defined by its IC50 value. This can be calculated for a given antagonist by determining the concentration of antagonist needed to elicit half inhibition of the maximum biological response of an agonist. Elucidating an IC50 value is useful for comparing the potency of drugs with similar efficacies; however the dose-response curves produced by both drug antagonists must be similar. The lower the IC-50, the greater the potency of the antagonist and the lower the concentration of drug that is required to inhibit the maximum biological response. Lower concentrations of drugs may be associated with fewer side-effects. The biological response is evaluated by determination of activation of downstream pathways linked to the VEGFR2, such as the c-Raf/MAPK, P13K/Akt and/or PLCyl/PKC pathway. The activation of said pathways can be measured by methods known to the person skilled in the art. In a preferred embodiment of the present invention the IC50 value of the antagonists for the VEGFR2 is below 50 μΜ, more preferably below 40 μΜ, such as below 30 μΜ, more preferably below 20 μΜ, such as below 10 μΜ, more preferably below 5 μΜ, such as below 2 μΜ, more preferably 1 μΜ, such as 0.5 μΜ, preferably 100nM, preferably below 90nM, such as below 80 nM, more preferably below 70 nM, such as below 60 nM, more preferably below 50 nM, such as below 40 nM, more preferably below 30 nM, such as below 20 nM, more preferably below 10 nM, such as below 5 nM, more preferably below 4 nM, such as below 3 nM, more preferably below 2 nM, such as below 1 nM, more preferably below 0.9 nM, such as below 0.8 nM, more preferably below 0.7 nM, such as below 0.6 nM, more preferably below 0.5 nM, such as below 0.4 nM, more preferably below 0.3 nM, such as below 0.2 nm, more preferably below 0.1 nM.
In a more preferred embodiment the IC50 value is below 1 nM, more preferably below 0.9 nM, such as below 0.8 nM, more preferably below 0.7 nM, such as below 0.6 nM, more preferably below 0.5 nM, such as below 0.4 nM, more preferably below 0.3 nM, such as below 0.2 nm, more preferably below 0.1 nM.
In the present invention the antagonist can be a competitive antagonist (also known as surmountable antagonist), which reversibly bind to receptors at the same binding site (active site) as the endogenous ligand or agonist, but without activating the receptor. Agonists and antagonists "compete" for the same binding site on the receptor. Once bound, an antagonist will block agonist binding. The level of activity of the receptor will be determined by the relative affinity of each molecule for the site and their relative concentrations. High concentrations of a competitive agonist will increase the proportion of receptors that the agonist occupies, and higher concentrations of the antagonist will be required to obtain the same degree of binding site occupancy. In another embodiment of the present invention the antagonist can be a non-competitive antagonist (sometimes called non-surmountable antagonist), which is an allosteric antagonist. These antagonists bind to a distinctly separate binding site from the agonist, exerting their action to that receptor via the other binding site. Non-competitive antagonists do not compete with agonists for binding. The bound antagonists may prevent conformational changes in the receptor required for receptor activation after the agonist binds. No amount of agonist can completely overcome the inhibition once it has been established. In functional assays of non-competitive antagonists, depression of the maximal response of agonist dose-response curves, and in some cases, rightward shifts, is produced. The rightward shift will occur as a result of a receptor reserve and inhibition of the agonist response will only occur when this reserve is depleted.
Uncompetitive antagonists differ from non-competitive antagonists in that they require receptor activation by an agonist before they can bind to a separate allosteric binding site. This type of antagonism produces a kinetic profile in which the same amount of antagonist blocks higher concentrations of agonist better than lower concentrations of agonist.
The antagonist of the present invention can be competitive, non-competitive and/or uncompetitive. Furthermore, the antagonists of the present invention can be biologically active peptide that binds to VEGFR2, an anti-VEGFR2 antibody and/or a biologically active molecule that binds to the tyrosine kinase domain of VEGFR2. In a preferred embodiment the antagonist of the present invention binds to the tyrosine kinase domain of VEGFR2 and/or to the kinase domain of a functional variant of VEGFR2 (as described in the section "VEGFR2").
In one embodiment of the present invention the VEGFR2 antagonist is of the formula (I):
Figure imgf000028_0001
Ri Formula (I)
Wherein,
X! is C or N
X2 is C or N
X3 is C or N
Figure imgf000028_0002
R2 is -O- CH3
R3 is -O- R8 R4 is -C- heteroaromatic ring structure
R5 is aromatic or heteroaromatic ring structure or ring system, optionally substituted with one or more substituent's selected from the group consisting of halogen, short alkyl, amide and - NH - C(O) - NH - R7
R6 is aromatic ring structure optionally substituted with halogen
R7 is heteroaromatic ring optionally substituted with short alkyl.
R8 is short alkyl optionally substituted with heterocycle, said heterocycle optionally substituted with short alkyl Preferably X3 is N, and when X2 is N, then X3 is also N. Preferably R5 is a benzyl ring. Short alkyl as used herein unless otherwise indicated is an alkyl chain of 1 -6 carbon atoms, preferably 1 -3 carbon atoms more preferably one carbon. In one embodiment of the invention the preferred short alkyl is methyl. In another embodiment of the present invention the VEGFR2 antagonist is of the formula (II):
Figure imgf000029_0001
Wherein,
X is N or C optionally substituted with R3
Ri is selected from the group consisting ofshort alkyl and alkenyl, optionally substituted with aromatic or heteroaromatic ring structure, said heteroaromatic ring structure optionally substituted with -C(O) - NH - C - C - NH(CH3)2
R2 is amide optionally substituted with aromatic or heteroaromatic ring structure optionally substituted with -NH-R4
R3 is selected from the group consisting of -OH and short alkyl.
R4 is aromatic ring optionally substituted with short alkyl or -S(0)2 - NH2 Preferably said heteroaromatic ring structure of R2 is a benzyl ring. In another embodiment of the present invention the VEGFR2 antagonist is of the formula (I I I):
Figure imgf000030_0001
Wherein,
Ri is an amide optionally substituted with aromatic or heteroaromatic ring structure, optionally substituted with halogen or triflourmethyl.
R2 is -O -R3.
R3 is an aromatic or heteroaromatic ring structure optionally substituted with - C(O) -
In a preferred embodiment of the present invention the VEGFR2 antagonist is selected from the group consisting of:
- N-(2-Chloro-4-((6,7-dimethoxy-4-quinolyl)oxy)phenyl)-N'-(5- methyl-3- isoxazolyl)urea
- N-Methyl-2-((3-((1 E)-2-(pyridin-2-yl)ethenyl)-1 H-indazol-6- yl)sulfanyl)benzamide (Axitinib)
- 4-((4-Fluoro-2-methyl-1 H-indol-5-yl)oxy)-6-methoxy-7-(3- (pyrrolidin-1 - yl)propoxy)quinazoline (Cediranib)
- N-(2-(Diethylamino)ethyl)-5-((Z)- (5-fluoro-1 ,2-dihydro-2-oxo-3H-indol-3- ylidene)methyl)-2,4- dimethyl- 1 H-pyrrole-3-carboxamide
- 5-((4-((2,3-Dimethyl-2H-indazol-6- yl)methylamino)-2-pyrimidinyl)amino)-2- methyl-benzenesulfonamide (Pazopanib)
- N-(4-Bromo-2-fluorophenyl)-6-methoxy-7-((1 -methyl-4- piperidinyl)methoxy)-4- quinazolinamine (Vandetanib)
- 4-(4-((((4-Chloro-3- (trifluoromethyl)phenyl)amino)carbonyl)amino)phenoxy)-N- methyl-2-pyridinecarboxamide
- N-(4-Chlorophenyl)-4-(4-pyridinylmethyl)-1 -phtalazinamine (Vatalanib)
- 3-((Z)-(3,5-Dimethylpyrrol-2-yl)methylene)-2-indolinone (Semaxanib), and
- SU1498 ((E)-3-(3,5-Diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl)amino- carbonyl]acrylonitrile) In another embodiment of the present invention the VEGFR2 antagonist is selected from the group consisting of:
- N-(2-Chloro-4-((6,7-dimethoxy-4-quinolyl)oxy)phenyl)-N'-(5- methyl-3- isoxazolyl)urea (AV-951 )
- N-Methyl-2-((3-((1 E)-2-(pyridin-2-yl)ethenyl)-1 H-indazol-6- yl)sulfanyl)benzamide (Axitinib)
- 4-((4-Fluoro-2-methyl-1 H-indol-5-yl)oxy)-6-methoxy-7-(3- (pyrrolidin-1 - yl)propoxy)quinazoline (Cediranib)
- N-(2-(Diethylamino)ethyl)-5-((Z)- (5-fluoro-1 ,2-dihydro-2-oxo-3H-indol-3- ylidene)methyl)-2,4- dimethyl- 1 H-pyrrole-3-carboxamide
- 5-((4-((2,3-Dimethyl-2H-indazol-6- yl)methylamino)-2-pyrimidinyl)amino)-2- methyl-benzenesulfonamide (Pazopanib)
- N-(4-Bromo-2-fluorophenyl)-6-methoxy-7-((1 -methyl-4- piperidinyl)methoxy)-4- quinazolinamine (Vandetanib)
- 4-(4-((((4-Chloro-3- (trifluoromethyl)phenyl)amino)carbonyl)amino)phenoxy)-N- methyl-2-pyridinecarboxamide
- N-(4-Chlorophenyl)-4-(4-pyridinylmethyl)-1 -phtalazinamine (Vatalanib)
- 3-((Z)-(3,5-Dimethylpyrrol-2-yl)methylene)-2-indolinone (Semaxanib),
- SU1498 (((E)-3-(3,5-Diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl)amino- carbonyl]acrylonitrile)
- (1 -{2-Chloro-4-[(6,7-dimethoxyquinolin-4-yl)oxy]phenyl}-3-(5-methylisoxazol-3- yl)urea) (Tivozanib)
- (17-Demethoxy-17-allylaminogeldanamycin) (Telatinib)
- (N-(2-diethylaminoethyl)-5-[(Z)-(5-fluoro-2-oxo-1 H-indol-3-ylidene)methyl]-2,4- dimethyl-1 H-pyrrole-3-carboxamide) (Sunitinib)
- (4-[4-[[4-chloro-3-(trifluoromethyl)phenyl]carbamoylamino]
phenoxy]-/V-methyl-pyridine-2-carboxamide) (Sorafenib)
- (4-[4-({[4-Chloro-3-(trifluoromethyl)phenyl]carbamoyl}amino)-3-fluorophenoxy]- A/-methylpyridine-2-carboxamide) (Regorafenib)
- RAF 265
- Puquitinib
- PF 337210
- Pegdinetanib - (5-[[4-[(2,3-Dimethyl-2H-indazol-6-yl)methylamino]-2-pyrimidinyl]amino]-2- methylbenzolsulfonamide) (Pazopanib)
- (/V-(4-irifluoromethoxyphenyl) 3~[(quinolin-4-ylmeihyi)amino]ihiophene-2"
carboxamide) (OS I 930)
- OSI 632
- (3-[2,4-dimethyl-5-[(Z)-(2-oxo-1 H-indol-3-ylidene)methyl]- 1 H-pyrrol-3-yl]propanoic acid) (Orantinib)
- (N-(3,3-Dimethyl-2,3-dihydro-1 H-indol-6-yl)-2-[(pyridin-4-lmethyl)amino]pyridine- 3-carboxamide) (Motesanib)
- ((gSJ OfiJ I fiJ Sfi^^J OJ ^^J S-Hexahydro-I O-methoxy-g-methyl-l l -
(methylamino)-9,13-epoxy-1 H,9H-diindolo[1 ,2,3-gh:3',2',1 '-lm]pyrrolo[3,4- j][1 ,7]benzodiamzonine-1 -one) (Midostaurin)
- Jl 101
- (methyl (3Z)-3-{[(4-{methyl[(4-methylpiperazin-1 - yl)acetyl]amino}phenyl)amino](phenyl)methylidene}-2-oxo-2,3-dihydro-1 H- indole-6-carboxylate) (Intedanib)
- (N-[3-Fiuoro-4-[[8-rrieihoxy-7-[3-(4-morpholinyi)propoxy3-4- quinol!nyl]oxy phenyl] Nl,-(4 luorophenyl)-1 ,1 -cyclopropanedicarboxamide) (Foretinib)
- (L-arginyl-L-phenylalanyl-L-valyl-L-prolyl-L-a-aspartylglycyl-
- L-asparaginyl-L-arginyl-L-isoleucine) (Elpamotide)
- E 7050
- E 3810
- CYC 1 16
- CEP 1 1981
- (4-[(4-fluoro-2-methyl-1 H-indol-5-yl)oxy]-6-methoxy-7-[3-(pyrrolidin-1 - yl)propoxy]quinazoline) (Cediranib)
- AEE 788
- (Λ/-[4-(1 -cyanocyclopentyl) phenyl-2-(4-picolyl)amino-3-nicotinamide
methanesulphonate) (Apatinib)
- (A/-Methyl-2-[[3-[(£)-2-pyridin-2-ylethenyl]-1 /-/-indazol-6-yl]sulfanyl]benzamide) (Axitinib)
- ((3R,4R)-4-amino-1 -((4-((3-methoxyphenyl)amino)pyrrolo[2,1 -f][1 ,2,4] triazin-5- yl)methyl)-3-piperidinol) (BMS 690514) - ((3R,4R)-4-amino-1 -((4-((3-methoxyphenyl)amino)pyrrolo[2J -f][1 ,2,4]triazin-5- yl)methyl)-3-piperidinol) (Brivanib alaninate)
- (A/-(4-((6J-Dimethoxyquinolin-4-yl)oxy)phenyl)-/V-(4-fluorophenyl)cyclopropane- 1 ,1 -dicarboxamide) (Cabozantinib)
- (A/-(4-chlorophenyl)-4-(pyridin-4-ylmethyl)phthalazin- 1 -amine) (Vatalanib)
In an even more preferred embodiment of the present invention the VEGFR2 antagonsit is selected from the group consisting of:
- N-Methyl-2-((3-((1 E)-2-(pyridin-2-yl)ethenyl)-1 H-indazol-6- yl)sulfanyl)benzamide (Axitinib)
- 4-((4-Fluoro-2-methyl-1 H-indol-5-yl)oxy)-6-methoxy-7-(3- (pyrrolidin-1 - yl)propoxy)quinazoline (Cediranib)
- N-(4-Bromo-2-fluorophenyl)-6-methoxy-7-((1 -methyl-4- piperidinyl)methoxy)-4- quinazolinamine (Vandetanib)
- 3-((Z)-(3,5-Dimethylpyrrol-2-yl)methylene)-2-indolinone (Semaxanib), and
- SU1498 ((E)-3-(3,5-Diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl)amino- carbonyl]acrylonitrile) In an even more preferred embodiment of the present invention the VEGFR2 antagonist is selected from the group consisting of:
- Axitinib
- Brivanib alaninate
- Pazopanib
- Sorafenib
- Sunitinib
- Tivozanib
- Vandetanib
- Cediranib
- Vatalanib
- SU1498 ((E)-3-(3,5-Diisopropyl-4-hydroxyphenyl)-2-[(3-phenyl-n-propyl)amino- carbonyl]acrylonitrile) In one embodiment the antagonist of the present invention is selected from the group consisting of ZM323881 , DC101 , MAB3572, AF357, GW654652, GW612286, GW695612, SU5416, SU1498, SU6668, SU5402, Src 11 , Pazopanib, Axtinib, Sorafenib, PTK787/ZK222584, AV951 , Cediranib, Sunitinib, and vandetanib, and other antagonists with same function and structure as these, including pro-drugs, crystals, solvates, salts and esters thereof. In one embodiment of the present invention the antagonist is a functional homologue of these antagonists.
In another embodiment the antagonist of the present invention is selected from the group consisting of ZM323881 , DC101 , MAB3572, AF357, GW654652, GW612286, GW695612, SU5416, SU1498, SU6668, SU5402, Src 11 , Pazopanib, Axtinib, Brivanib alaninate, Pazopanib, Sorafenib, PTK787/ZK222584, AV951 , Cediranib, Sunitinib, Tivozanib, Vatalanib and vandetanib, and other antagonists with same function and structure as these, including pro-drugs, crystals, solvates, salts and esters thereof. In one embodiment of the present invention the antagonist is a functional homologue of these antagonists.
Other VEGFR inhibitors described in the following patents and patent applications can be used in the present invention: US 6,563,618, US 2003/016601 1 , US 2006/0223133, PCT/JP 1998/05697, US 2006/0241 1 15, WO 2005/070891 , US 6,258,812, US 2003/0105091 , WO 01 /37820, US 6,235,764, WO 01 /32651 , US 6,630,500, US 6,515,004, US 6,713,485, US 5,521 ,184, US 5,770,599, US 5,747,498, WO 02/68406, WO 02/66470, WO 02/55501 , WO 04/05279, WO 04/07481 , WO 04/07458, WO 06/012374, WO 06/1 16713, WO 04/09784, WO 02/591 10, WO 99/45009, WO 00/59509, WO 99/61422, US 5,990,141 , WO 00/12089 and WO 00/02871 each of which is herein incorporated by reference in its entirety, particularly in parts disclosing VEGFR inhibitors.
In one embodiment the VEGFR2 antagonist is an anti-VEGFR2 antibody. In another embodiment the molecule binding the cancer stem-like cells according to the present invention is an antibody. Said antibody can be any suitable antibody known in the art. In one embodiment the antagonist of the present invention is a binding partner such as immunologically active fragments of antibodies or single chain antibodies. Antibody molecules are typically Y-shaped molecules whose basic unit consists of four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked by disulfide bonds. Each of these chains is folded in discrete domains. The C-terminal regions of both the heavy and light chains are conserved in sequence and are called the constant regions, also known as C-domains. The N-terminal regions, also known as V-domains, are variable in sequence and are responsible for the antibody specificity. The antibody specifically recognizes and binds to an antigen mainly through six short complementary-determining regions located in their V- domains.
In one embodiment of the invention the VEGFR2 antagonist is a binding partner, such as a fragment of an anti-VEGFR2 antibody, preferably an antigen binding fragment or variable region. Examples of antibody fragments useful with the present invention include Fab, Fab', F(ab')2 and Fv fragments. Papain digestion of antibodies produces two identical antigen bindig fragments, called the Fab fragment, each with a single antigen binding site, and a residual "Fc" fragment, so-called for its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen binding fragments which are capable of cross-linking antigen, and a residual other fragment (which is termed pFc').
Additional fragments can include diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.
The antibody fragments Fab, Fv and scFv differ from whole antibodies in that the antibody fragments carry only a single antigen-binding site. Recombinant fragments with two binding sites have been made in several ways, for example, by chemical cross-linking of cysteine residues introduced at the C-terminus of the VH of a Fv (Cumber et al., 1992), or at the C-terminus of the VL of an scFv (Pack and Pluckthun, 1992), or through the hinge cysteine residues of Fab's (Carter et al., 1992).
Preferred antibody fragments retain some or essential all the ability of an antibody to selectively binding with its antigen or receptor. Preferred fragments of the present invention are defined as follows:
Fab is the fragment that contains a monovalent antigen-binding fragment of an antibody molecule. A Fab fragment can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain. Fab' is the fragment of an antibody molecule and can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain. Two Fab' fragments are obtained per antibody molecule.
Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxyl terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region.
(Fab')2 is the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction. F(ab')2 is a dimer of two
Fab' fragments held together by two disulfide bonds. Fv is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy and one light chain variable domain in a tight, non- covalent association (VH -V L dimer). It is in this configuration that the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH -V L dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site.
In one embodiment of the present invention the binding partner is a single chain antibody ("SCA"), defined as a genetically engineered molecule comprising the variable region of the light chain, and the variable region of the heavy chain, linked by a suitable poly-5 peptide linker as a genetically fused single chain molecule. In one embodiment the SCA consists of only part of the variable region of the light chain, and part of the variable region of the heavy chain, linked by a suitable poly-5 peptide linker as a genetically fused single chain molecule. In a preferred embodiment the SCA consists of the variable region of the light chain, and the variable region of the heavy chain, linked by a suitable poly-5 peptide linker as a genetically fused single chain molecule. Such single chain antibodies are also referred to as "single-chain Fv" or "sFv" antibody fragments. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the sFv to form the desired structure for antigen binding.
The antibody fragments to be used with the invention may be produced in any suitable manner known to the person skilled in the art. Several microbial expression systems have already been developed for producing active antibody fragments, e.g. the production of Fab in various hosts, such as E. coli, yeast, and the filamentous fungus Trichoderma reesei are known in the art. The recombinant protein yields in these alternative systems can be relatively high. 1 -2 g/l for Fab secreted to the periplasmic space of E. coli in high cell density fermentation or at a lower level, e.g. about 0.1 mg/l for Fab in yeast in fermenters, and 150 mg/l for a fusion protein CBHI-Fab and 1 mg/l for Fab in Trichoderma in fermenters and such production is very cheap compared to whole antibody production in mammalian cells (hybridoma, myeloma, CHO).
The fragments can be produced as Fab's or as Fv's, but additionally it has been shown that a VH and a VL can be genetically linked in either order by a flexible polypeptide linker, which combination is known as an scFv.
In one embodiment of the present invention the antibody is preferably selected from the group consisting of a monoclonal antibody, a fragment of an antibody, a derivative of an antibody, a chimerized antibody, a humanized antibody and a single chain antibody. Said antibody of the present invention should function as a VEGFR2 antagonist and demonstrate the same preferred IC50 values as described above in this section. In one embodiment the antagonist of the present invention is a NRP-1 antagonist. The NRP-1 antagonist may be a small molecule, a peptide and/or a protein and may be selected from the group consisting of EG01257, Anti-neuropilin-1 , Anti-NRP1 , M 1685A, M1685A, MNRP 1685A, MNRP1685A, R 7347, R7347 and RG7347, and other antagonists with same function and structure as these, including pro-drugs, crystals, solvates, salts and esters thereof. In one embodiment of the present invention the antagonist is a functional homologue of these antagonists.
In one embodiment of the present invention the NRP-1 antagonist is of the formula (IV):
Figure imgf000038_0001
or a pharmaceutically acceptable salt thereof,
wherein:
W is arylene, heteroarylene or Formula (V)
Figure imgf000038_0002
Formula (V); each L is independently alkylene, alkenylene, alkynylene, a direct bond, arylene, cycloalkylene, alkylene-arylene, alkylene-C=0 or-C=0; preferably C1 -C6 alkylene, C1 - C6 alkenylene, C5-C6 arylene, C3-C6 cycloalkylene, C1 -C6 alkylene-arylene or c1 -c6 alkylene-C=0;
each X is independently an N-containing heteroarylene, N-containing cycloalkylene or NR;
Y is N-containing heteroaryl, N-containing cycloalkyl, NR2, OR1 , CN or C02R;
Z1 is
Figure imgf000038_0003
R is H or C1 -C6 alkyl;
R1 is H, C1 -C6 alkyl or an amino acid;
n is 2, 3, 4 or 5; and
m is 1 , 2 or 3.
In another embodiment of the present invention the NRP-1 antagonist is of the formula
Figure imgf000039_0001
Formula (VI) or a pharmaceutically acceptable salt thereof,
wherein:
each L is independently alkylene, alkenylene, alkynylene, a direct bond, arylene, cycloalkylene, alkylene-arylene, or alkylene-C=0; preferably C1 -C6 alkylene, C1 -C6 alkenylene, C5-C6 arylene, C3-C6 cycloalkylene, C1 -C6 alkylene-arylene or c1 -c6 alkylene-C=0;
each X is independently an N-containing heteroarylene, N-containing cycloalkylene or NR;
Y is N-containing heteroaryl, N-containing cycloalkyl, NR, OR1 , CN or C02R;
Z1 is ;
Figure imgf000040_0001
R is H or C1 -C6 alkyl;
R1 is H, C1 -C6 alkyl or an amino acid;
n is O, 1 , 2, 3, 4 or 5; and
m is 1 , 2 or 3.
In a preferred embodiment the NRP-1 antagonist is selected from the group consisting of EG01257, Anti-neuropilin-1 , Anti-NRP1 , M 1685A, M1685A, MNRP 1685A, MNRP1685A, R 7347, R7347 and RG7347.
Labelling
In one embodiment of the invention the molecule that specifically binds VEGFR2 (preferably a VEGFR2 antagonist, such as a VEGFR2 antibody) or a synthetic probe is directly labelled with a detectable label, wherein the detectable label preferably may be selected from the group consisting of fluorochromes, colour agents, heavy metals, enzymes and radioactive labels. Said labels are preferably covalently attached thereto. Preferably the VEGFR2 antagonist of the present invention is labelled with fluorochromes. In one embodiment of the invention the antagonist described in the section "antagonist" are labelled. Said antagonist may be used in the present invention as described in the section "method of detecting cancer stem-like cells and/or progenitor cells". The binding of such probes or antagonists to VEGFR2 on or in the cell may be observed under a fluorescence microscope as a bright fluorescence or may be detected by a fluorimetric apparatus. Instead of direct labelling or in addition to the direct labelling in another embodiment the probes or antagonists are indirectly labelled with biotin or other binding partners or enzymes for example, such as alkaline phosphatase or peroxidase. In the present invention a binding partner is a molecule which is binding the VEGFR2 antagonist with high affinity or is linked to the VEGFR2 antagonist, and therefore can be used for detection of the binding of the VEGFR2 antagonist to VEGFR2. In a preferred embodiment the antagonist is indirectly labelled with biotin. Biotin may be detected using suitable streptavid in/avid in molecules known to people skilled in the art. These complexes may comprise fluorochromes or suitable enzymes. By use of a combination of labelling methods it is possible to enhance the signals from the cancer stem-like cells, thereby facilitating the identification thereof as described in the section "method of detecting a cancer stem-like cell and/or progenitor cells".
In order to enhance the probability and/or selectivity of identifying the cancer stem-like cells over the background, two or more selective labellings may be performed. The two or more labellings may be a combination of any of the labellings used for single labelling described above. Accordingly, the combined labelling may be carried out by the use of two or more different hybridisation probes. Likewise a combination of different antagonists may be used, either with specificity for the same antigen or with specificity for different antigens. In further embodiments labelling with a combination of nucleotide probes and antagonists may be performed. In one embodiment a combination of a labelled VEGFR2 antagonist as described in the section "antagonist" and a labelled antibody for another antigen other than VEGFR2 may be used as described in the section "method of detecting a cancer stem-like cell and/or progenitor cells".
By use of suitable techniques known in the art, the selectively labelled cells may be isolated from the cell sample and processed further. The isolated cancer stem-like cells and/or progenitor cells expressing VEGFR2 may be used for diagnosis or as marker of the presence of cancer stem-like cells and/or progenitor cells expressing VEGFR2 as described in the section "method of detecting a cancer stem-like cell and/or progenitor cells".
The invention also relates to embodiments wherein other molecules than VEGFR2 antagonists are labelled with a detectable label, e.g. molecules specifically binding CD31 , Tusc5, 7B4, BNH9/BNF13, CD105, CD146, D2-40, EN4, PAL-E, CD133 or integrin Alpha-6. These molecules may also be labelled with any of the detectable labels described in this section.
Radiation therapy
The present invention relates in one embodiment to the combination of administration of a VEGFR2 antagonist and/or a NRP-1 antagonist as described in the section "antagonist" and radiation therapy. Radiation therapy, also denoted radiotherapy and/or radiation oncology, and sometimes abbreviated to XRT, according to the present invention is the medical use of ionizing radiation (IR) as part of cancer treatment to control malignant cells (not to be confused with radiology, the use of radiation in medical imaging and diagnosis). Radiation therapy may be used for curative or adjuvant treatment. It may be used as palliative treatment (where cure is not possible and the aim is for local disease control or symptomatic relief) or as therapeutic treatment (where the therapy has survival benefit and it can be curative).
Radiotherapy may be used for the treatment of tumours (benign as well as malignant), and may be used as a primary or adjuvant modality. It is also common to combine radiotherapy with surgery, cytotoxic drugs, hormone therapy or some mixture of the three. Most common tumour types can be treated with radiotherapy in some way. The precise treatment intent (curative, adjuvant, neoadjuvant, therapeutic, or palliative) will depend on the tumour type, location, and stage, as well as the general health of the patient.
To spare normal tissues (such as skin or organs which radiation must pass through in order to treat the tumour), shaped radiation beams may in the present invention be aimed from several angles of exposure to intersect at the tumour, providing a much larger absorbed dose there than in the surrounding, healthy tissue.
Radiation therapy works by damaging the DNA of cells. The damage is caused by a photon, electron, proton, neutron, or ion beam directly or indirectly ionizing the atoms which make up the DNA chain. Indirect ionization happens as a result of the ionization of water, forming free radicals, notably hydroxyl radicals, which then damage the DNA. In the most common forms of radiation therapy, most of the radiation effect is through free radicals. Because cells have mechanisms for repairing DNA damage, breaking the DNA on both strands proves to be the most significant technique in modifying cell characteristics. Because cancer cells generally are undifferentiated and stem cell-like, they reproduce more, and have a diminished ability to repair sub-lethal damage compared to most healthy differentiated cells. The DNA damage is inherited through cell division, accumulating damage to the cancer cells, causing them to die or reproduce more slowly.
In the present invention the amount of radiation used in radiation therapy is measured in gray (Gy), and varies depending on the type and stage of cancer being treated. In one embodiment of the present invention radiation therapy is given for curative cases, and the typical dose for a solid epithelial tumour ranges from 60 to 80 Gy, while lymphomas are treated with 20 to 40 Gy. In another embodiment of the present invention radiation therapy is administered for preventative (adjuvant) purposes in which case the doses are typically around 45 - 60 Gy in 1 .8 - 2 Gy fractions (for Breast, Head and Neck cancers respectively.) Many other factors are considered by radiation oncologists when selecting a dose, including whether the patient is receiving cytotoxic drugs, patient comorbidities, whether radiation therapy is being administered before or after surgery, and the degree of success of surgery.
In the present invention the amount of radiation used is preferably in the ranges 80-100 Gy, more preferably 60-80 Gy, such as 60-70 Gy, more preferably 40-60 Gy, such as 40-50 Gy, more preferably 20-40 Gy, such as 20-30 Gy, more preferably 1 -20 Gy, such as 1 -10 Gy, more preferably 1 -2 Gy.
Depending on the radiation delivery method, several angles or sources may be used to sum to the total necessary dose. The total dose may be fractionated (spread out over time) for several important reasons. Fractionation allows normal cells time to recover, while tumour cells are generally less efficient in repair between fractions. Fractionation may also allow tumour cells that were in a relatively radio-resistant phase of the cell cycle during one treatment to cycle into a sensitive phase of the cycle before the next fraction is given. Similarly, tumour cells that were chronically or acutely hypoxic (and therefore more radioresistant) may reoxygenate between fractions, improving the tumour cell kill. In one embodiment of the present invention, two fractions of radiation therapy per day are used near the end of a course of treatment. This schedule, known as a concomitant boost regimen or hyperfractionation, is used on tumours that regenerate more quickly when they are smaller. In particular, tumours in the head-and- neck demonstrate this behavior.
One of the best-known alternative fractionation schedules is Continuous Hyperfractionated Accelerated Radiotherapy (CHART). CHART, used to treat lung cancer, consists of three smaller fractions per day.
In the present invention the fractionation schedule may preferably consists of 1 .8 to 2 Gy per day, for 3 to 7, preferably five days a week, more preferably 1 .5 to 1 .8 Gy per day, for 3 to 7, preferably five days a week.
As part of the present the position of the radiation source can differ; external beam radiotherapy positions the radiation source outside the body, brachytherapy uses sealed radioactive sources placed precisely in the area under treatment, and systemic radioisotopes are given by infusion or oral ingestion. In the present invention brachytherapy can use temporary or permanent placement of radioactive sources. The temporary sources are usually placed by a technique called afterloading. In afterloading a hollow tube or applicator is placed surgically in the organ to be treated, and the sources are loaded into the applicator after the applicator is implanted. In one embodiment of the present invention Intraoperative radiotherapy or IORT is used, which is a special type of radiotherapy that is delivered immediately after surgical removal of the cancer. This method has been employed in breast cancer (TARGeted Introperative radioTherapy or TARGIT), brain tumours and rectal cancers.
In the present invention any of the abovementioned radiation therapies are contemplated to be part of the invention. In the present invention radiation therapy may be used curative and palliative and in combination with the administration of a VEGFR2 antagonist as described in the section "antagonist".
In one embodiment of the present invention a VEGFR2 antagonist, as described in the section "antagonist", is administered immediately prior to the administration of radiation therapy, as described here above. Immediately prior to radiation therapy as used herein, unless otherwise indicated, denotes an administration 5 days of initiation of radiation therapy, more preferably with 4 days, such as within 3 days, more preferably within 2 days, such as with in 1 day, more preferably within 20 hours, such as within 10 hours, more preferably within 5 hours, more preferably within 4.5 hours, such as with in 4 hours, more preferably within 3.5 hours, such as within 3 hours, more preferably within 2.5 hours, such as within 2 hours, more preferably within 1 .5 hours, such as within 1 hour, more preferably within 0.5 hour, such as within 15 minutes, more preferably within 10 minutes, such as within 5 minutes of radiation therapy. In a preferred embodiment the VEGFR2 antagonist is administered within 2 hour of initiation of radiation therapy, such as within 1 hour, more preferably with 0.5 hour of initiation of radiation therapy.
In another embodiment of the present invention a VEGFR2 antagonist, as described in the section "antagonist", is administered prior to the administration of radiation therapy, as described here above. Prior to radiation therapy as used herein, unless otherwise indicated, denotes an administration within 3 weeks of initiation of radiation therapy, more preferably within 2 weeks, such as within 1 week, more preferably within 6 days, such as within 5 days, more preferably with 4 days, such as within 3 days, more preferably within 2 days, such as with in 1 day, more preferably within 20 hours, such as within 10 hours, more preferably within 5 hours, more preferably within 4.5 hours, such as with in 4 hours, more preferably within 3.5 hours, such as within 3 hours, more preferably within 2.5 hours, such as within 2 hours, more preferably within 1 .5 hours, such as within 1 hour, more preferably within 0.5 hour, such as within 15 minutes, more preferably within 10 minutes, such as within 5 minutes of radiation therapy. In a preferred embodiment the VEGFR2 antagonist is administered within 2 hour of initiation of radiation therapy, such as within 1 hour, more preferably with 0.5 hour of initiation of radiation therapy. In another embodiment of the present invention a NRP-1 antagonist, as described in the section "antagonist", is administered immediately prior to the administration of radiation therapy, as described here above. Immediately prior to radiation therapy as used herein, unless otherwise indicated, denotes an administration within 3 weeks of initiation of radiation therapy, more preferably within 2 weeks, such as within 1 week, more preferably within 6 days, such as within 5 days of initiation of radiation therapy, more preferably with 4 days, such as within 3 days, more preferably within 2 days, such as with in 1 day, more preferably within 20 hours, such as within 10 hours, more preferably within 5 hours, more preferably within 4.5 hours, such as with in 4 hours, more preferably within 3.5 hours, such as within 3 hours, more preferably within 2.5 hours, such as within 2 hours, more preferably within 1 .5 hours, such as within 1 hour, more preferably within 0.5 hour, such as within 15 minutes, more preferably within 10 minutes, such as within 5 minutes of radiation therapy. In a preferred embodiment the NRP-1 antagonist is administered within 2 hour of initiation of radiation therapy, such as within 1 hour, more preferably with 0.5 hour of initiation of radiation therapy.
In another embodiment of the present invention a NRP-1 antagonist, as described in the section "antagonist", is administered in combination with an anti-VEGF antibody (e.g. avastin) immediately prior to the administration of radiation therapy, as described here above. Immediately prior to radiation therapy as used herein, unless otherwise indicated, denotes an administration within 3 weeks of initiation of radiation therapy, more preferably within 2 weeks, such as within 1 week, more preferably within 6 days, such as within 5 days of initiation of radiation therapy, more preferably with 4 days, such as within 3 days, more preferably within 2 days, such as with in 1 day, more preferably within 20 hours, such as within 10 hours, more preferably within 5 hours, more preferably within 4.5 hours, such as with in 4 hours, more preferably within 3.5 hours, such as within 3 hours, more preferably within 2.5 hours, such as within 2 hours, more preferably within 1 .5 hours, such as within 1 hour, more preferably within 0.5 hour, such as within 15 minutes, more preferably within 10 minutes, such as within 5 minutes of radiation therapy. In a preferred embodiment the NRP-1 antagonist is administered within 2 hour of initiation of radiation therapy, such as within 1 hour, more preferably with 0.5 hour of initiation of radiation therapy.
Hyperproliferative diseases
The hyperproliferative disease to be treated or reducing the risk of recurrence of with the present invention is contemplated to be any disease originating in hyperproliferative cells present in the subject to be treated. In one embodiment the hyperproliferative disease is a tumour of connective tissue origin, nervous system origin, and/or hematologic origin. A tumour or tumor is the name for a neoplasm or a solid lesion formed by an abnormal growth of cells (termed neoplastic) which looks like a swelling. Tumour is not synonymous with cancer. A tumour can be benign, pre-malignant or malignant, whereas cancer is by definition malignant. In the present apllication the words tumour and tumor are used interchangeable.
In another embodiment the hyperproliferative disease is a cancer of connective tissue origin, nervous system origin, and/or hematologic origin. A cancer (medical term: malignant neoplasm) is a class of diseases in which a group of cells display uncontrolled growth (division beyond the normal limits), invasion (intrusion on and destruction of adjacent tissues), and sometimes metastasis (spread to other locations in the body via lymph or blood). These three malignant properties of cancers differentiate them from benign tumours, which are self-limited, and do not invade or metastasize. Most cancers form a tumour but some, like leukemia, do not. In one embodiment the disease to be treated, prevented or reducing the risk of recurrence of is ovarian and/or prostate cancer. In a preferred embodiment the disease to be treated, prevented or reducing the risk of recurrence of is a tumour of connective tissue origin, nervous system origin, and/or hematologic origin. In a more preferred embodiment the disease to be treated, prevented or reducing the risk of recurrence of is a cancer of connective tissue origin, nervous system origin, and/or hematologic origin. In a more preferred embodiment the disease to be treated, prevented or reducing the risk of recurrence of is selected from the group consisting of head and neck tumours, Kaposi's sarcoma, CNS neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas, cerebral metastases, melanomas, gastrointestinal sarcomas, renal sarcomas, rhabdomyosarcomas, gliomas, glioblastomas, glioblastoma multiforme, leimyosarcomas, squamous cell carcinomas, basal cell carcinomas, human malignant keratinocytes, leukemias, multiple myelomas, lymphomas, acute myelocytic leukemia (AML), chronic myelocytic leukemia (CLL), erythrocytic leukemia, monocytic leukemia, Hodgkin's disease and Non-Hodgkin's disease.
In a more preferred embodiment the disease to be treated, prevented or reducing the risk of recurrence of is selected from the group consisting of glioma, glioblastoma, glioblastoma multiforme, astrocytoma, oligodendroglioma, ependymomas, medulloblastoma, meningioma, neurinoma and Schwannoma.
In an even more preferred embodiment the disease to be treated, prevented or reducing the risk of recurrence of is glioblastomas or glioblastoma multiforme.
Pharmaceutical compositions and Administration
In one aspect of this invention, there is provided a pharmaceutical composition comprising, as an active ingredient, a VEGFR2 antagonist of the present invention together with a pharmaceutically acceptable carrier or diluent. The antagonist of the invention may be administered alone or in combination with pharmaceutically acceptable carriers, diluents or excipients, in either single or multiple doses. Suitable pharmaceutical acceptable carriers, diluents and excipients include inert solid diluents or fillers, sterile aqueous solutions and various organic solvents.
The pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 21 st Edition, 2000, Lippincott Williams & Wilkins.
The pharmaceutical compositions formed by combining a VEGFR2 antagonist, or a pharmaceutically acceptable salt, solvate or prodrug thereof, with pharmaceutical acceptable carriers, diluents or excipients can be readily administered in a variety of dosage forms such as tablets, powders, lozenges, syrups, suppositories, injectable solutions and the like. In powders, the carrier is a finely divided solid such as talc or starch which is in a mixture with the finely divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired. Suitable solid carriers include, but are not limited to, lactose, terra alba, sucrose, cyclodextrins (such as hydroxypropyl- -cyclodextrin, HPCD), talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Examples of liquid carriers include, but are not limited to, syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, polysorbates (such as Tween-20 or Tween-80), Cremophor EL or water. Similarly, the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax. Also contemplated are nano- formulations, such as nano-emulsion or nano-dispersions. In a preferred embodiment of the invention the excipients used in the pharmaceutical formulation conforms to the "Generally recognized as Safe" GRAS listing provided by the FDA.
The pharmaceutical compositions may be specifically formulated for administration by any suitable route such as the oral and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intradermal) route or local injection. It will be appreciated that the preferred route will depend on the general condition and age of the subject to be treated, the nature of the condition to be treated and the active ingredient chosen.
Pharmaceutical compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders and granules. Where appropriate, they can be prepared with coatings such as enteric coatings or they can be formulated so as to provide controlled release of the active ingredient such as sustained or prolonged release according to methods well known in the art.
For oral administration in the form of a tablet or capsule, a VEGFR2 antagonist of the present invention may suitably be combined with an oral, non-toxic, pharmaceutically acceptable carrier such as ethanol, glycerol, water or the like. Furthermore, suitable binders, lubricants, disintegrating agents, flavouring agents and colourants may be added to the mixture, as appropriate. Suitable binders include, e.g., lactose, glucose, starch, gelatin, acacia gum, tragacanth gum, sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes or the like. Lubricants include, e.g., sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride or the like. Disintegrating agents include, e.g., starch, methyl cellulose, agar, bentonite, xanthan gum, sodium starch glycolate, crospovidone, croscarmellose sodium or the like. Additional excipients for capsules include macrogols or lipids. For the preparation of solid compositions such as tablets, the active compound of formula (I) is mixed with one or more excipients, such as the ones described above, and other pharmaceutical diluents such as water to make a solid preformulation composition containing a homogenous mixture of a compound of the present invention. The term "homogenous" is understood to mean that the compound of the present invention is dispersed evenly throughout the composition so that the composition may readily be subdivided into equally effective unit dosage forms such as tablets or capsules. The preformulation composition may then be subdivided into unit dosage forms containing for example from about 0.05 to about 1000 mg, in particular from about 0.1 to about 500 mg, e.g. 10-200 mg, such as 30-180 mg, such as 20-50 mg of the active compound of the invention. In the form of a dosage unit, the compound may be administered one or more times a day at appropriate intervals, always depending, however, on the frequency and mode of administration, the sex, age, weight and general condition of the subject treated, the nature and severity of the condition treated and any concomitant diseases to be treated and other factors evident to those skilled in the art. Conveniently, a dosage unit of a formulation contain between 0.1 mg and 1000 mg, preferably between 1 mg and 100 mg, such as 5-50 mg of a compound of the invention.
Liquid formulations for either oral or parenteral administration of the VEGFR2 antagonist include, e.g., aqueous solutions, syrups, elixirs, aqueous or oil suspensions and emulsion with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut oil. Suitable dispersing or suspending agents for aqueous suspensions include synthetic or natural gums such as tragacanth, alginate, acacia, dextran, sodium carboxymethylcellulose, gelatin, methylcellulose or polyvinylpyrolidone.
Pharmaceutical compositions for parenteral administration include sterile aqueous and non-aqueous injectable solutions, dispersions, suspensions or emulsions as well as sterile powders to be reconstituted in sterile injectable solutions or dispersions prior to use. For parenteral administration, solutions containing a compound of this invention or a pharmaceutically acceptable salt, solvate or prodrug thereof in sesame or peanut oil, aqueous propylene glycol, or in sterile aqueous solution may be employed. Such aqueous solutions should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. The oily solutions are suitable for intra-articular, intra-muscular and subcutaneous injection purposes. The preparation of all these solutions under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
It is preferred to use parenteral administration for VEGFR2 antagonist of the invention, wherein the active part of the molecule contains acid labile groups, such as e.g. ester groups. By using parenteral administration the acidic environment of the stomach is avoided together with the first-pass metabolism.
Depot injectable formulations are also contemplated as being within the scope of the present invention.
In addition to the aforementioned ingredients, the formulations of a compound of the present invention may include one or more additional ingredients such as diluents, buffers, flavouring agents, colourant, surface active agents, thickeners, preservatives, e.g. methyl hydroxybenzoate (including anti-oxidants), emulsifying agents and the like. A suitable dosage of the compound of the invention will depend on the age and condition of the patient, the severity of the disease to be treated and other factors well known to the practicing physician. The compound may be administered for example either orally, parenterally or topically according to different dosing schedules, e.g. daily or with intervals, such as weekly intervals. In general a single dose will be in the range from 0.01 to 100 mg/kg body weight, preferably from about 0.05 to 75 mg/kg body weight, more preferably between 0.1 to 50 mg/kg body weight, and most preferably between 0.1 to 25 mg/kg body weight. The compound may be administered as a bolus (i.e. the entire daily dosis is administered at once) or in divided doses two or more times a day. Variations based on the aforementioned dosage ranges may be made by a physician of ordinary skill taking into account known considerations such as weight, age, and condition of the person being treated, the severity of the affliction, and the particular route of administration.
The VEGFR2 antagonists of this invention are generally utilized as the free substance or as a pharmaceutically acceptable salt or ester thereof. One example is an acid addition salt of a compound having the utility of a free base. When a VEGFR2 antagonist contains a free base such salts are prepared in a conventional manner by treating a solution or suspension of a free base of the antagonist with a chemical equivalent of a pharmaceutically acceptable acid, for example, inorganic and organic acids. Physiologically acceptable salts of a compound with a hydroxy group include the anion of said compound in combination with a suitable cation such as sodium or ammonium ion.
If the treatment involves administration of another therapeutically active compound it is recommended to consult Goodman & Gilman's The Pharmacological Basis of Therapeutics, 1 1 th Ed., McGraw-Hill 2005, for useful dosages of said compounds. The administration of a VEGFR2 antagonist of the present invention with one or more other active compounds may be either concomitantly or sequentially. In one embodiment the other active compound may be a NRP-1 antagonist.
The VEGFR2 antagonist of the invention may also be formulated in a pharmaceutical composition comprising one or more further active substances alone, or in combination with pharmaceutically acceptable carriers, diluents, or excipients in either single or multiple doses. The suitable pharmaceutical acceptable carriers, diluents and excipients are as described herein above, and the one or more further active substances may be any active substances, or preferably an active substance as described in the section "combination treatment" herein below.
In the present invention the preferred administration is parenteral and/or oral. In one embodiment the VEGFR2 antagonist is administered directly to the tumour site. In one embodiment administration directly to the tumour site is done through a gliadel wafer. Gliadel wafers for use in the present invention are small, dime-sized biodegradable polymer wafers that are designed to deliver VEGFR2 antagonist directly into the surgical cavity created when a brain tumour is resected. Gliadel wafers are commercially available and can be purchased from Eisai Inc. (www.gliadel.com).
Combination treatment.
A compound of the present invention may also be used to advantage in combination with one or more other anti-proliferative or anti-neoplastic agents. Such antiproliferative agents include, but are not limited to aromatase inhibitors; antiestrogens; topoisomerase I inhibitors; topoisomerase II inhibitors; microtubule active agents; alkylating agents; histone deacetylase inhibitors; compounds which induce cell differentiation processes; cyclooxygenase inhibitors; MMP inhibitors; mTOR inhibitors; antineoplastic antimetabolites; platin compounds; compounds targeting/decreasing a protein or lipid kinase activity and further anti-angiogenic compounds; compounds which target, decrease or inhibit the activity of a protein or lipid phosphatase; gonadorelin agonists; anti-androgens; angiostatic steroids; methionine aminopeptidase inhibitors; bisphosphonates; biological response modifiers; antiproliferative antibodies; heparanase inhibitors; inhibitors of Ras oncogenic isoforms; telomerase inhibitors; proteasome inhibitors; agents used in the treatment of hematologic malignancies; compounds which target, decrease or inhibit the activity of Flt-3; Hsp90 inhibitors; NRP-1 antagonist; temozolomide (TEMOD AL(R)); leucovorin; immune stimulating agents, such as BCG, IL-2 or IFN-a , antibodies, such as rituximab or herceptin and cancer vaccines.
A compound of the present invention may also be used to advantage in combination with known therapeutic processes, e.g., the administration of hormones or tumour cell damaging approaches, especially ionizing radiation.
A compound of the present invention may also be used as a radiosensitizer, including, for example, the treatment of tumours which exhibit poor sensitivity to radiotherapy. A compound of the present invention may also be given in combination with one or more radiosensitizing drugs. A radiosensitizing drug used in the present invention is a drug which makes tumours more sensitive to radiation therapy. Such radiosensitizing drugs include but are not limited to; Cisplatin, Nimorazole and Cetuximab. In one embodiment of the present invention the VEGFR2 antagonist is administered in combination with Cisplatin and/or Nimorazole and/or Cetuximab. The VEGFR2 antagonist and the radiosensitizing drug may be administered simultaneously or sequential as a combined medicament or as discrete entities. In one embodiment the radiosensitizing drug and the VEGFR2 antagonist is formulated as a combined medicament. By the term "combination", is meant either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where a compound of the present invention and a combination partner may be administered independently at the same time or separately within time intervals that especially allow that the combination partners show a cooperative, e.g., synergistic, effect, or any combination thereof. Method of treatment
In a further aspect the present invention relates to a method of treating or preventing diseases in a subject, said method comprises administering to said subject a therapeutically effective amount of a VEGFR2 antagonist, or pharmaceutically acceptable salts, solvates or prodrugs thereof, as defined herein, to a subject in need of such treatment. The disease may be any disease or disorder as mentioned herein, such as for example mentioned in the section "hyperproliferative diseases", and the VEGFR2 antagonist may be administered alone or in a pharmaceutical composition, such as for example mentioned in the section "Pharmaceutical compositions". In the present invention preventing the disease is linked to reducing the risk of recurrence as described above. By reducing the risk of recurrence of the disease, the disease is in one embodiment prevented. When no disease has yet been observed in the subject, the administration of VEGFR2 antagonist according to the present invention can prevent the occurrence of the disease.
In a preferred embodiment of this aspect of the invention the method is a method of treating or preventing a hyperproliferative disease in a subject, said method comprises administering to said subject a therapeutically effective amount of a VEGFR2 antagonist of the present invention, or pharmaceutically acceptable salts, solvates or prodrugs thereof, as defined herein, to a subject in need of such treatment. The hyperproliferative disease may be any hyperproloferative disease as described herein above. Preferably the hyperproliferative disease is glioblastoma or glioblastoma multiforme. In one embodiment of the method of treatment or prevention of a hyperproliferative disease according to the invention, the VEGFR2 antagonist of the present invention, or pharmaceutically acceptable salts, solvates or prodrugs thereof, as defined herein, is administered in combination with one or more further active substances. The active substances may be any active substances, and preferably an active substance as described herein above in the section "combination treatment". More preferably the one or more additional active substances are selected from the group consisting of antiproliferative, anti-neoplastic agents or radiosensitizing drugs.
In one embodiment of the method of treatment or prevention of a hyperproliferative disease according to the invention, the VEGFR2 antagonist of the present invention, or pharmaceutically acceptable salts, solvates or prodrugs thereof, as defined herein, is administered in combination with ionizing radiation therapy. The radiation therapy may be any form of radiation, and preferably ionizing radiation as described herein above in the section "radiation therapy".
In a preferred embodiment the compound of the present invention is administered to a subject immediately prior to radiation therapy.
In one embodiment the method of treatment or prevention of a hyperproliferative disease according to the invention, comprises administration of the VEGFR2 antagonist of the present invention, or pharmaceutically acceptable salts, solvates or prodrugs thereof, as defined herein, in combination with administration of VEGF antibodies. In a preferred embodiment the compound of the present invention is administered to a subject prior to administration of anti-VEGF antibodies. The method of treatment or prevention of a hyperproliferative disease according to the invention may consist of multiple treatments administered to the same subject.
Examples
Example 1
Cell isolation and culture. Glioma cell populations enriched or depleted in cancer stem-like cells were isolated from human glioma surgical specimens. Briefly, tumours were immediately dissected with removal of gross necrosis; washed in Earle's balanced salt solution; subjected to a papain digestion followed by trituration, filtering, and lysis of RBC in PBS/water (1 :3) solution; and then cultured overnight (12 h) in neurobasal medium (with B27 and epidermal growth factor and basic fibroblast growth factor at 20 ng/mL) for recovery of cellular surface antigens before cell sorting. Primary glioma tumour samples were obtained from patients undergoing resection.
Fluorescence-activated cell sorting analysis. Because papain digestion may cause loss of some cellular surface antigens such as CD133, it is critical to allow the isolated total tumour cells to reexpress their surface antigens in the neurobasal medium overnight (12 h) before sorting for CD133+ glioma cells. Tumour cell cultures were subjected to fluorescence-activated cell sorting (FACS) analysis and cell sorting after the antigen recovery period. Human-specific anti-VEGFR2 (Biolegend) conjugated to AlexaFluor 647 conjugate was used for cell sorting and FACS analysis. In vivo tumour formation assays. VEGFR2 positive (top 10%) and negative (bottom 10%) glioma cells were transplanted into athymic BalbC nu/nu mice through intracranial or s.c. injection (figure 7).
Neurosphere formation efficiency. To determine the effect of VEGFR2 expression on the ability of glioma stem-like cells to form neurospheres, disaggregated cells were labeled with VEGFR2 AlexaFluor 647 antibody and sorted at a density of 1 cell per well into 96-well plates via a flow cytometry cell sorter (MoFlow XDP, Beckman Coulter). The percentage of wells with neurospheres was determined on day 10.
Immunofluorescent staining. Frozen human sections from glioblastoma multiforme were fixed with 4% paraformaldehyde, washed with TBS, incubated with anti-VEGFR2 monoclonal antibody (AbCam) and rabbit CD133 (AbCam) or CD31 (AbCam), and FITC-conjugated donkey anti-mouse IgG or anti-rabbit secondary antibody. Cells were co-stained with 4',6-diamidino-2-phenylindole (DAPI) and mounted with the anti-fade medium. Stained cells were examined under a fluorescent microscope (Zeiss Axiovert 200). Cell growth curves and inhibitor testing. Cells were aliquoted into a 96-well plate at 1 ,000 or 4000 cells per well in duplicate. Cell growth was measured at the day 1 , 3, 5 and 7, and the effect of inhibitor was measured at 24, 48, 72, 96 hours after SU1498 or SU5416 treatment using the CellTiter-Glo assay kit (Promega, Madison, Wl, http://www.promega.com).
Ionizing radiation treatment in enrichment experiments of freshly dissociated human specimens and xenografts. Freshly isolated ells were treated with ionizing radiation of 8 Gy (dose rate 2.18 Gy/min by an X-ray generator, Pantak, Berkshire, United Kingdom; HF160; 150 kV; 15mA) after 1 hour pre-treatment with the inhibitor SU1498 or left untreated, and analyzed 24, 48 and 72 hours after irradiation for the presence of CD133- and VEGFR2-positive cells using FACS Calibur (BD Biosciences).
Example 2
In vivo experiments in mice. Experiment 1 : Injection of freshly dissociated cells from human biopsy or mouse xenograft that will have VEGFR2-knockdown using lentiviral shRNA. Two groups of mice will be injected:
A) non-targeting shRNA - 10000 cells intracranial^
B) VEGFR2-targeting shRNA - 10000 cells intracranial^
Experiment 2: Intracranial injection of 100 000 VEGFR2 negative cells and 5, or 8 days later, mice will be sacrificed and brain will be analyzed for presence of human VEGFR2 expressing cell - this is in order to evaluate re-expression of VEGFR2 due to stimuli from microenvironment.
Experiment 3: To show that radiation does not efficiently kill VEGFR2 positive cells, and so IR treatment should follow anti-VEGFR2 therapy: Mice will be divided into 4 groups:
a) injected with VEGFR2 negative cells
b) injected with VEGFR2 positive cells
c) injected with VEGFR2 shRNA cells
d) injected with VEGFR2 non-targeting shRNA
Mice will be treated with equal doses of IR and tumour formation will be followed. We expect mice with VEGFRshRNA to be significantly delayed in tumour formation.
Sequence listing:
SEQ ID NO: - VEGFR2 ) iild-type
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIVVVVG YRIYDVVLSP SHGIELSVGE KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE RVISFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I I LVG TAVIAMFFWL LLVI ILRTVK RANGGELKTG YLS IVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 2 - Natural variant of VEGFR2 substituted at position 2
MRSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD
WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD
YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD
SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIVVVVG YRIYDVVLSP SHGIELSVGE
KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS
DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP
EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP
PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY
PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE
RVISFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT
PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I I LVG TAVIAMFFWL LLVI ILRTVK RANGGELKTG YLS IVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 3 - Natural variant of VEGFR2 substituted at position 136
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD
WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD
YRSPFIASVS DQHGVMYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD
SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIVVVVG YRIYDVVLSP SHGIELSVGE
KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS
DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP
EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP
PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY
PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE
RVISFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT
PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT
VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR
NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I I LVG TAVIAMFFWL
LLVI ILRTVK RANGGELKTG YLS IVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL
GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN
LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK
RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA
SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR
VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML
DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS
CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS
GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS
SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV SEQ ID NO: 4 - Natural variant of VEGFR2 substituted at position 248
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD
WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD
YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD
SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIWWG YRIYDVVLSP SHGIELSVGE
KLVLNCTGRT ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS
DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP
EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP
PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY
PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE
RVI SFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT
PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT
VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR
NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I I LVG TAVIAMFFWL
LLVI ILRTVK RANGGELKTG YLS IVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL
GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN
LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK
RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA
SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR
VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML
DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS
CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS
GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS
SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 5 - Natural variant of VEGFR2 substituted at position 275
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD
WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD
YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD
SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIWWG YRIYDVVLSP SHGIELSVGE
KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNLDLKTQ SGSEMKKFLS TLTIDGVTRS
DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP
EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP
PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY
PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE
RVI SFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT
PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT
VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I I LVG TAVIAMFFWL LLVI ILRTVK RANGGELKTG YLS IVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 6 - Natural variant Ooff VEGFR2 substituted at position 297
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIVVVVG YRIYDVVLSP SHGIELSVGE KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGITRS DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP EIKWYKNGIP LESNHTIKAG HVLT IIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE RVI SFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT PVCKNLDTLW KLNATMFSNS TNDI LLIIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I I LVG TAVIAMFFWL LLVI ILRTVK RANGGELKTG YLS IVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNL SSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 7 - Natural variant of VEGFR2 substituted at position 462
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIVVVVG YRIYDVVLSP SHGIELSVGE KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QVEEECANEP SQAVSVTNPY PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE RVI SFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I I LVG TAVIAMFFWL LLVI ILRTVK RANGGELKTG YLS IVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 8 - Natural variant of VEGFR2 substituted at position 472
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD
WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD
YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD
SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIVVVVG YRIYDVVLSP SHGIELSVGE
KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS
DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP
EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP
PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SHAVSVTNPY
PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE
RVI SFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT
PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT
VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR
NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I I LVG TAVIAMFFWL
LLVI ILRTVK RANGGELKTG YLS IVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL
GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 9 - Natural variant of VEGFR2 substituted at position 482
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD
WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD
YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD
SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIWWG YRIYDVVLSP SHGIELSVGE
KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS
DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP
EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP
PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY
PREEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE
RVI SFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT
PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT
VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR
NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I I LVG TAVIAMFFWL
LLVI ILRTVK RANGGELKTG YLS IVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL
GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN
LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK
RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA
SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR
VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML
DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS
CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS
GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS
SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 10 - Natural variant of VEGFR2 substituted at position 539
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD
WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD
YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD
SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIWWG YRIYDVVLSP SHGIELSVGE KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRRE RVISFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I ILVG TAVIAMFFWL LLVI ILRTVK RANGGELKTG YLSIVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 1 1 - Natural variant of VEGFR2 substituted at position 689
MQSKVLLAVA LWLCVETRAA SVGL SVSLD LSIQKDI LTIKANTTLQ ITCRGQRDLD WLWPNNQSGS EQRVEVTECS DGLF KTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD YRSPFIASVS DQHGVVYITE NKNK VVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD SKKGFTIPSY MISYAGMVFC EAKINDESYQ SIM"YIVVVVG YRIYDVVLSP SHGIELSVGE KLVLNCTART ELNVGIDFNW EYPSSKHQHK NRDLKTQ SGSEMKKFLS TLTIDGVTRS DQGLYTCAAS SGLMTKKNST FVRVHEKPFV SGMESLV EATVGERVRI PAKYLGYPPP EIKWYKNGIP LESNHTIKAG HVLTIMEVSE GNYTVIL TNPISKEKQS HVVSLVVYVP PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI HHIHWYW QLEEECANEP SQAVSVTNPY PCEEWRSVED FQGGNKIEVN KNQF.ALIEGK VSTLVIQ AANVSALYKC EAVNKVGRGE RVISFHVTRG PEITLQPDMQ PTEQESVSLW DRSTFEN LTWYKLGPQP LPIHVGELPT PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT VLERVAPTIT GNLENQTTSI GESIEVSCMA SGN:PPPQIMW FKDNETLVED SGIVLKDGNR NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I ILVG TAVIAMFFWL LLVI ILRTVK RANGGELKTG YLSTVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL GRGAFGQVIE ADAFGIDKTA TCRT'VAVKML KEG.ATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRN:EFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 12 - Natural variant Of VEGFR2 substituted at position 814
MQSKVLLAVA LWLCVETRAA SVGL: 'SLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD WLWPNNQSGS EQRVEVTECS DGLF 'LTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD YRSPFIASVS DQHGVVYITE NKNK 'IPC LGSISNLNVS LCARYPEKRF VPDGNRISWD SKKGFTIPSY MISYAGMVFC ΕΑΚΓ ;SYQ SIMYIVVVVG YRIYDVVLSP SHGIELSVGE KLVLNCTART ELNVGIDFNW EYPS [QHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS DQGLYTCAAS SGLMTKKNST FVRV :PFV AFGSGMESLV EATVGERVRI PAKYLGYPPP EIKWYKNGIP LESNHTIKAG HVLT I ;VSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP PQIGEKSLIS PVDSYQYGTT QTLT c1 ΎΑΙ PPPHHIHWYW QLEEECANEP SQAVSVTNPY PCEEWRSVED FQGGNKIEVN KNQF. ALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE RVISFHVTRG PEITLQPDMQ PTEQE: S 'SLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT PVCKNLDTLW KLNATMFSNS TNDILI IELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT VLERVAPTIT GNLENQTTSI GESI ;CTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR NLTIRRVRKE DEGLYTCQAC SVLG' :VEA FFI IEGAQEK TNLEI I ILVG TAVIAMFFWL LLVI ILRTVK RANGGELKTG YLSI' )PDE LPLNEHCERL PYDASKWEFP RDRLKLGKPL GRGAFGQEIE ADAFGIDKTA TCRT" 'KML KEGATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNL S 'LRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLS ;EEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKI 'GLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGAS 'GVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLL [AQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYL1 ;KRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGG: 'SKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQ: D ;GTT LSSPPV
SEQ ID NO: 13 - Natural variant of VEGFR2 substituted at position 848
MQSKVLLAVA LWLCVETRAA SVGL SVSLD LSIQKDI LTIKANTTLQ ITCRGQRDLD WLWPNNQSGS EQRVEVTECS DGLF KTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD YRSPFIASVS DQHGVVYITE NKNK VVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD SKKGFTIPSY MISYAGMVFC EAKINDESYQ SIM"YIVVVVG YRIYDVVLSP SHGIELSVGE KLVLNCTART ELNVGIDFNW EYPSSKHQHK NRDLKTQ SGSEMKKFLS TLTIDGVTRS DQGLYTCAAS SGLMTKKNST FVRVHEKPFV SGMESLV EATVGERVRI PAKYLGYPPP EIKWYKNGIP LESNHTIKAG HVLTIMEVSE GNYTVIL TNPISKEKQS HVVSLVVYVP PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE RVISFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I ILVG TAVIAMFFWL LLVI ILRTVK RANGGELKTG YLSIVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL GRGAFGQEIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 14 - Natural variant of VEGFR2 substituted at position 873
MQSKVLLAVA LWLCVETRAA SVGL SVSLD LSIQKDI LTIKANTTLQ ITCRGQRDLD WLWPNNQSGS EQRVEVTECS DGLF KTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD YRSPFIASVS DQHGVVYITE NKNK VVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD SKKGFTIPSY MISYAGMVFC EAKINDESYQ SIM"YIVVVVG YRIYDVVLSP SHGIELSVGE KLVLNCTART ELNVGIDFNW EYPSSKHQHK NRDLKTQ SGSEMKKFLS TLTIDGVTRS DQGLYTCAAS SGLMTKKNST FVRVHEKPFV SGMESLV EATVGERVRI PAKYLGYPPP EIKWYKNGIP LESNHTIKAG HVLTIMEVSE GNYTVIL TNPISKEKQS HVVSLVVYVP PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI HHIHWYW QLEEECANEP SQAVSVTNPY PCEEWRSVED FQGGNKIEVN KNQF.ALIEGK VSTLVIQ AANVSALYKC EAVNKVGRGE RVISFHVTRG PEITLQPDMQ PTEQESVSLW DRSTFEN LTWYKLGPQP LPIHVGELPT PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT VLERVAPTIT GNLENQTTSI GESIEVSCTA SGN:PPPQIMW FKDNETLVED SGIVLKDGNR NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I ILVG TAVIAMFFWL LLVI ILRTVK RANGGELKTG YLSI"VMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL GRGAFGQVIE ADAFGIDKTA TCRT'VAVKML KE ATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRN:EFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGAS IDE:EFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLL DYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SOYL KRK VSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 15 - Natural variant of VEGFR2 substituted at position 952
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD
WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD
YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD
SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIWWG YRIYDVVLSP SHGIELSVGE
KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS
DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP
EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP
PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY
PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE
RVI SFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT
PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT
VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR
NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFI IEGAQEK TNLEI I I LVG TAVIAMFFWL
LLVI ILRTVK RANGGELKTG YLS IVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL
GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN
LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YIGAIPVDLK
RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA
SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDARLPLK WMAPETIFDR
VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML
DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS
CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS
GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS
SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV
SEQ ID NO: 16 - Natural variant of VEGFR2 substituted at position 1065
MQSKVLLAVA LWLCVETRAA SVGLPSVSLD LPRLSIQKDI LTIKANTTLQ ITCRGQRDLD
WLWPNNQSGS EQRVEVTECS DGLFCKTLTI PKVIGNDTGA YKCFYRETDL ASVIYVYVQD
YRSPFIASVS DQHGVVYITE NKNKTVVIPC LGSISNLNVS LCARYPEKRF VPDGNRISWD
SKKGFTIPSY MI SYAGMVFC EAKINDESYQ SIMYIWWG YRIYDVVLSP SHGIELSVGE
KLVLNCTART ELNVGIDFNW EYPSSKHQHK KLVNRDLKTQ SGSEMKKFLS TLTIDGVTRS
DQGLYTCAAS SGLMTKKNST FVRVHEKPFV AFGSGMESLV EATVGERVRI PAKYLGYPPP
EIKWYKNGIP LESNHTIKAG HVLTIMEVSE RDTGNYTVIL TNPISKEKQS HVVSLVVYVP
PQIGEKSLIS PVDSYQYGTT QTLTCTVYAI PPPHHIHWYW QLEEECANEP SQAVSVTNPY
PCEEWRSVED FQGGNKIEVN KNQFALIEGK NKTVSTLVIQ AANVSALYKC EAVNKVGRGE
RVI SFHVTRG PEITLQPDMQ PTEQESVSLW CTADRSTFEN LTWYKLGPQP LPIHVGELPT PVCKNLDTLW KLNATMFSNS TNDILIMELK NASLQDQGDY VCLAQDRKTK KRHCVVRQLT VLERVAPTIT GNLENQTTSI GESIEVSCTA SGNPPPQIMW FKDNETLVED SGIVLKDGNR NLTIRRVRKE DEGLYTCQAC SVLGCAKVEA FFIIEGAQEK TNLEIIILVG TAVIAMFFWL LLVI ILRTVK RANGGELKTG YLS IVMDPDE LPLDEHCERL PYDASKWEFP RDRLKLGKPL GRGAFGQVIE ADAFGIDKTA TCRTVAVKML KEGATHSEHR ALMSELKILI HIGHHLNVVN LLGACTKPGG PLMVIVEFCK FGNLSTYLRS KRNEFVPYKT KGARFRQGKD YVGAIPVDLK RRLDSITSSQ SSASSGFVEE KSLSDVEEEE APEDLYKDFL TLEHLICYSF QVAKGMEFLA SRKCIHRDLA ARNILLSEKN VVKICDFGLA RDIYKDPDYV RKGDTRLPLK WMAPETIFDR VYTIQSDVWS FGVLLWEIFS LGASPYPGVK IDEEFCRRLK EGTRMRAPDY TTPEMYQTML DCWHGEPSQR PTFSELVEHL GNLLQANAQQ DGKDYIVLPI SETLSMEEDS GLSLPTSPVS CMEEEEVCDP KFHYDNTAGI SQYLQNSKRK SRPVSVKTFE DIPLEEPEVK VIPDDNQTDS GMVLASEELK TLEDRTKLSP SFGGMVPSKS RESVASEGSN QTSGYQSGYH SDDTDTTVYS SEEAELLKLI EIGVQTGSTA QILQPDSGTT LSSPPV

Claims

Claims
A pharmaceutical composition comprising a therapeutically effective amount of a vascular endothelial growth factor receptor 2 (VEGFR2) antagonist for treatment of a tumour selected from the group consisting of connective tissue tumours, tumours of the nervous system, hematologic tumours, lymphoid leukemias and/or myeloid leukemias in a subject in need thereof, wherein the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy.
A pharmaceutical composition comprising a therapeutically effective amount of a vascular endothelial growth factor receptor 2 (VEGFR2) antagonist for reducing the risk of recurrence of a hyperproliferative disease in a subject in need thereof by inhibiting growth of cancer stem-like cells and/or progenitor cells.
The composition according to claim 2, wherein the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy.
The composition according to claims 2-3, wherein the hyperproliferative disease is a tumour of connective tissue origin, nervous system origin, and/or hematologic origin.
The composition according to claims 2-3, wherein the hyperproliferative disease is a cancer of connective tissue origin, nervous system origin, and/or hematologic origin.
The pharmaceutical composition according to any preceding claim, comprising a therapeutically effective amount of a vascular endothelial growth factor receptor 2 (VEGFR2) antagonist for treatment of connective tissue tumours, tumours of the nervous system, hematologic tumours, lymphoid leukemias and/or myeloid leukemias, and reducing the risk of recurrence of said hyperproliferative disease, in a subject in need thereof, wherein the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy.
7. The composition according to any preceding claim, wherein the tumour is
selected from the group consisting of head and neck tumours, Kaposi's sarcoma, CNS neoplasms, neuroblastomas, capillary hemangioblastomas, meningiomas, cerebral metastases, melanomas, gastrointestinal sarcomas, renal sarcomas, rhabdomyosarcomas, gliomas, glioblastomas, glioblastoma multiforme, leimyosarcomas, squamous cell carcinomas, basal cell carcinomas, human malignant keratinocytes, leukemias, multiple myelomas, lymphomas, acute myelocytic leukemia (AML), chronic myelocytic leukemia (CLL), erythrocytic leukemia, monocytic leukemia, Hodgkin's disease and Non- Hodgkin's disease.
8. The compostition according to any preceding claim, wherein the tumour is
selected from the group consisting of glioma, glioblastoma, glioblastoma multiforme, astrocytoma, oligodendroglioma, ependymomas, medulloblastoma, meningioma, neurinoma and Schwannoma.
9. The composition according to any preceding claims, wherein the tumour is glioblastomas or glioblastoma multiforme.
10. The composition according to any preceding claim, wherein the antagonist is a biologically active peptide that binds to VEGFR2.
1 1 . The composition according to claim 10, wherein the antagonist is selected from the group consisting of SU1498 and ZM323881 .
12. The composition according to any preceding claim, wherein the antagonist is an anti-VEGFR2 antibody.
13. The composition according to claim 12, wherein the anti-VEGFR2 antibody is selected from the group consisting of a monoclonal antibody, a fragment of an antibody, a derivative of an antibody, a chimerized antibody, a humanized antibody and a single chain antibody.
14. The composition according to claim 12, wherein the anti-VEGFR2 antibody is selected from the group consisting of DC101 , MAB3572 and AF357.
15. The composition according to any preceding claims, wherein the antagonist binds to the tyrosine kinase domain of VEGFR2.
16. The composition according to claim 1 -13, wherein the antagonist inhibits the stability of VEGFR2.
17. The composition according to claim 15, wherein the antagonist is selected from the group consisting of GW654652, GW612286, GW695612, SU5416, SU1498, SU6668, SU5402, Src 11 , Pazopanib, Axtinib, Sorafenib and
PTK787/ZK222584.
18. The composition according to any preceding claim, wherein the antagonist has an IC50 towards the VEGFR2 below 50 μΜ, more preferably below 40 μΜ, such as below 30 μΜ, more preferably below 20 μΜ, such as below 10 μΜ, more preferably below 5 μΜ, such as below 2 μΜ, more preferably 1 μΜ, such as 0.5 μΜ, more preferably 10OnM, preferably below 90nM, such as below 80 nM, more preferably below 70 nM, such as below 60 nM, more preferably below 50 nM, such as below 40 nM, more preferably below 30 nM, such as below 20 nM, more preferably below 10 nM, such as below 5 nM, more preferably below 4 nM, such as below 3 nM, more preferably below 2 nM, such as below 1 nM, more preferably below 0.9 nM, such as below 0.8 nM, more preferably below
0.7 nM, such as below 0.6 nM, more preferably below 0.5 nM, such as below 0.4 nM, more preferably below 0.3 nM, such as below 0.2 nm, more preferably below 0.1 nM.
19. The composition according to any preceding claim, wherein the antagonist is administered orally, parenterally and/or locally to the tumour.
20. The composition according to any preceding claim, wherein the antagonist is administered using a gliadel wafer placed directly in the surgical cavity created after removal of a brain tumour.
21 . The composition according to any preceding claim, wherein a radiosensitizing drug is administered together with said composition.
22. The composition according to any preceding claim, wherein the radiosensitizing drug is selected from the group consisting of Cisplatin, Nimorazole, Cetuximab, Temozolomide and PARP inhibitors.
23. The composition according to any preceding claim, wherein said composition is administered within 2 days of initiation of radiation therapy, such as with in 1 day, more preferably within 20 hours, such as within 10 hours, more preferably within 5 hours, more preferably within 4.5 hours, such as with in 4 hours, more preferably within 3.5 hours, such as within 3 hours, more preferably within 2.5 hours, such as within 2 hours, more preferably with 1 .5 hours, such as within 1 hour.
24. The composition according to any preceding claim, wherein said antagonist is able to cross the cell membrane of mammalian cells and the blood-brain barrier of the subject.
25. The composition according to any preceding claim, wherein the subject is
mammalian, more preferable primate, more preferably human.
26. The composition according to any of the preceding claims, wherein the
treatment is curative, ameliorating or palliative.
27. A pharmaceutical composition comprising a therapeutically effective amount of a Neuropilin-1 (NRP-1 ) antagonist for treatment of a tumour selected from the group consisting of connective tissue tumours, tumours of the nervous system, hematologic tumours, lymphoid leukemias and/or myeloid leukemias in a subject in need thereof, wherein the pharmaceutical composition is prepared for administration to said subject immediately prior to radiation therapy.
28. The composition according to claim 27 wherein the composition is administered in combination with the VEGFR2 antagonist of claim 10-18.
29. The composition according to claim 27, wherein the composition is
administered in combination with an anti-VEGF antibody.
30. A method of treating a tumour in a subject by inhibiting the cancer stem-like cells and/or progenitor cells comprising:
a. administering to the subject a therapeutically effective amount of a vascular endothelial growth factor receptor 2 (VEGFR2) antagonist, and b. immediately after subjecting the tumour in the subject to ionizing
radiation therapy.
31 . A method of reducing the risk of recurrence of a tumour in a subject by
inhibiting the cancer stem-like cells and/or progenitor cells comprising:
a. administering to the subject a therapeutically effective amount a
vascular endothelial growth factor receptor 2 (VEGFR2) antagonist, and b. immediately thereafter subjecting said tumour to ionizing radiation
therapy.
32. A method of treating a tumour in a subject by inhibiting the cancer stem-like cells and/or progenitor cells comprising:
a. administering to the subject a therapeutically effective amount of a Neuropilin-1 (NRP-1 ) antagonist, and
b. immediately after subjecting the tumour in the subject to ionizing
radiation therapy.
33. The method of claim 32, wherein the NRP-1 antagonist is administered in combination with the VEGFR2 antagonist of claim 10-18.
34. The method of claim 32, wherein the NRP-1 antagonist is administered in combination with an anti-VEGF antibody.
35. A method of reducing the risk of recurrence of a tumour in a subject by
inhibiting the cancer stem-like cells and/or progenitor cells comprising:
a. administering to the subject a therapeutically effective amount a
Neuropilin-1 (NRP-1 ) antagonist, and b. immediately thereafter subjecting said tumour to ionizing radiation therapy.
36. The method of claim 35, wherein the NRP-1 antagonist is administered in
combination with the VEGFR2 antagonist of claim 10-18.
37. The method of claim 35, wherein the NRP-1 antagonist is administered in
combination with an anti-VEGF antibody.
38. The method according to claims 27-37, wherein a radiosensitizing drug is
administered together with said antagonist.
39. The method according to claims 30-31 , wherein the antagonist is a biologically active peptide that binds to VEGFR2.
40. The method according to claims 30-31 , wherein the antagonist is an anti- VEGFR2 antibody
41 . The method according to claims 30-31 , wherein the antagonist binds to the tyrosine kinase domain of VEGFR2.
42. The method according to claims 30-31 and 32, wherein step a) is repeated multiple times prior to performing step b)
43. The method according to claims 30-42, wherein said antagonist is able to cross the cell membrane of mammalian cells and the blood-brain barrier of the subject.
44. A method of identifying a compound capable of inhibiting cancer stem-like cells and/or progenitor cells through binding to VEGFR2 said method comprising contacting VEGFR2
a. with a test compound, and
b. detecting the autophosphorylation of VEGFR2 and/or activation of c- Raf/MAPK, P13K/Akt and/or PLCyl/PKC pathway, and c. selecting the compounds which inhibits an activation of said pathways described in (b)
45. The method according to claim 44, wherein the VEGFR2 is expressed on cells or cell lines expressing VEGFR2, comprising contacting said cells
a. with a test compound, and
b. detecting the activation of c-Raf/MAPK, P13K/Akt and/or PLCyl/PKC pathway, and
c. selecting the compounds which inhibits an activation of said pathways described in (b)
46. The method according to claim 45, wherein the cell or cell line is selected from the group consisting of human astroglial tumour cell lines U1 18MG, U87MG, T98G, A172, Gli-06, SF126, U373MG, U251 MG, primary cells from
Glioblastoma Multiforme, primary cells from patients diagnosed with cancer, primary cell lines and primary xenograft cells.
47. A method according to claim 44, wherein the VEGFR2 is linked to a reporter gene, comprising contacting said VEGFR2
a. with a test compound, and
b. detecting the activation of said reporter gene, and
c. selecting the compounds which inhibits an activation of said reporter gene described in (b)
48. The method according to claims 44-45 and 47, wherein the detection of
activation is done by Western blot analysis.
49. A method for isolation of cancer stem-like cells and/or progenitor cells
comprising
a. contacting the population of cells with a molecule that specifically binds VEGFR2, and
b. isolating cells that bind the molecule that specifically binds VEGFR2, and
c. removing any endothel cells.
50. The method according to claim 49, wherein the population of cells is contacted with CD133 and/or integrin Alpha-6.
51 . The method according to claims 49-50, wherein the population of cells is
contacted with CD31 .
52. The method of claim 49, wherein the population of cells are isolated from a tumour, preferably a tumour of the nervous system.
53. The method according to any preceding claim, wherein the tumour is selected from the group consisting of glioma, glioblastoma, glioblastoma multiforme, astrocytoma, oligodendroglioma, ependymomas, medulloblastoma, meningioma, neurinoma and Schwannoma.
54. The method according to any preceding claims, wherein the tumour is
glioblastomas or glioblastoma multiforme.
55. A pharmaceutical composition comprising a compound identified by the method according to claims 44-45 and/or 47 for reducing the risk of recurrence of a hyperproliferative disease in a subject in need thereof by inhibiting growth of cancer stem-like cells.
56. A method for determining the presence of cancer stem-like cells and/or
progenitor cells in a tumour from a subject comprising a. contacting the cells of said tumour with a molecule that binds VEGFR2 b. detecting the presence of VEGFR2 in said cells.
57. The method according to claim 56, wherein said subject is mammalian, more preferable primate, more preferably human.
58. The method according to claims 56-57, wherein the population of cells is
contacted with CD133 and/or integrin Alpha-6.
59. The method according to claims 56-58, wherein the population of cells is contacted with CD31 .
60. A method for evaluating the efficiency of tumour treatment schedule of a subject by detecting the presence of VEGFR2 in cells isolated from said tumour, after said subject has undergone anti-tumour treatment.
61 . The method of claim 60, wherein said subject is mammalian, more preferable primate, more preferably human.
62. The method according to claims 60-61 , wherein the population of cells is
contacted with CD133 and/or integrin Alpha-6.
63. The method according to claims 60-62, wherein the population of cells is
contacted with CD31 .
64. A method for treatment comprising the steps of
i. determining the presence of cancer stem-like cells and/or progenitor cells in a tumour from a subject by detecting the presence of VEGFR2 in cells isolated from said tumour, and ii. evaluating whether the cells isolated from said tumour expresses VEGFR2, and
iii. administering to said subject a therapeutically effective amount of a VEGFR2 antagonist for treatment of a VEGFR2 positive tumour as evaluated in step ii,
wherein the administration to said subject is done immediately prior to ionizing radiation therapy.
65. The method according to claim 64, wherein said subject is mammalian, more preferable primate, more preferably human.
66. The method according to claims 64-65, wherein the population of cells is
contacted with CD133 and/or integrin Alpha-6. The method according to claims 64-66, wherein the population of cells is contacted with CD31 .
A method of identifying a subject sensitive to the treatment of claim 30-43 comprising the steps of: i. determining the presence of cancer stem-like cells and/or progenitor cells in a tumour from a subject by detecting the presence of VEGFR2 in cells isolated from said tumour, and ii. evaluating whether the cells isolated from said tumour expresses VEGFR2.
A method of determining the presence of internalized VEGFR2 in a cell comprising the steps of: i. determining the presence of internalized VEGFR2 in paraffin- embedded sections of a tumour from a subject by immunohistochemistry staining of said paraffin-embedded section using a primary conjugated VEGFR2 antibody, and ii. washing out non-bound VEGFR2 antibody, and
iii. fixating and permeabilizing the cells of said paraffin-embedded section, and
iv. staining said paraffin-embedded section again using the same VEGFR2 antibody, and
v. evaluating whether the cells present in the paraffin-embedded section expresses intracellular VEGFR2.
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